Chromatography systems and methods using them

ABSTRACT

Certain embodiments described herein are directed to chromatography systems that include a microfluidic device. The microfluidic device can be fluidically coupled to a switching valve to provide for selective control of fluid flow in the chromatography system. In some examples, the microfluidic device may include a charging chamber, a bypass restrictor or other features that can provide for added control of the fluid flow in the system. Methods of using the devices and methods of calculating lengths and diameters to provide a desired flow rate are also described.

PRIORITY APPLICATIONS

This application claims priority to U.S. application Ser. No. 12/472,948filed on Mar. 27, 2009. U.S. application Ser. No. 12/472,948 claimedpriority to each of U.S. Provisional Patent Application No. 61/056,225filed on May 27, 2008, to U.S. Provisional Patent Application No.61/142,702 filed on Jan. 6, 2009, to U.S. Provisional Patent ApplicationNo. 61/142,705 filed on Jan. 6, 2009, to U.S. Provisional PatentApplication No. 61/158,001 filed on Mar. 6, 2009 and to U.S. ProvisionalPatent Application No. 61/179,028 filed on May 18, 2009, the entiredisclosure of each of which is hereby incorporated herein by referencein its entirety.

TECHNOLOGICAL FIELD

Certain features, aspect and embodiments are directed to gaschromatography systems. In particular, certain embodiments are directedto chromatography systems that include a microfluidic device to controlfluid flow to one or more other components in the system.

BACKGROUND

Separations of complex samples can be difficult with existingchromatography systems. In particular, samples having peaks that eluteclosely can be difficult to separate. In addition, there may also be aneed for backflushing, heartcutting, column switching and detectorswitching.

SUMMARY

In one aspect, a chromatography system comprising a first injectorfluidically coupled to a first column, a second injector fluidicallycoupled to a second column, a microfluidic device fluidically coupled tothe first column and the second column, the microfluidic devicecomprising an internal microchannel fluidically coupled to a switchingvalve, and a detector fluidically coupled to the microfluidic device andconfigured to receive effluent from the first column when the switchingvalve is actuated to a first position and to receive effluent from asecond column when the switching valve is actuated to a second positionis provided.

In certain embodiments, the microfluidic device can include a first portfluidically coupled to the first column and a second port fluidicallycoupled to the second column. In certain examples, the detector is amass spectrometer. In some examples, the system can include a seconddetector fluidically coupled to the microfluidic device. In certainembodiments, the system can also include a modulating gas sourcefluidically coupled to the microfluidic device. In some examples, theinternal microchannel comprises a first charging chamber. In certainembodiments, the switching valve permits fluid flow from the modulatinggas source to the first charging chamber in a first position andrestricts fluid flow from the modulating gas source to the firstcharging chamber in a second position. In certain examples, the systemcan further include a second charging chamber in the microchannel. Insome embodiments, the switching valve permits fluid flow from themodulating gas source to the first charging chamber in a first positionand restricts fluid flow from the modulating gas source to the firstcharging chamber in a second position and permits fluid flow from themodulating gas source to the second charging chamber in the secondposition. In certain embodiments, the system may include a restrictorbetween the detector and the microfluidic device to balance fluid flowin the system.

In another aspect, a chromatography system comprising an injectorfluidically coupled to a first column, a microfluidic device fluidicallycoupled to the first column, the microfluidic device fluidically coupledto a switching valve through a port on the microfluidic device, a secondcolumn fluidically coupled to the microfluidic device, and a firstdetector fluidically coupled to the second column is disclosed.

In certain embodiments, the system can further include a second detectorfluidically coupled to the microfluidic device through a restrictorbetween the microfluidic device and the second detector. In certainexamples, the system can further include a second detector fluidicallycoupled to the microfluidic device through a third column between themicrofluidic device and the second detector. In some examples, thesystem may further include a restrictor between the second column andthe first detector. In other examples, the microfluidic device comprisesan internal bypass restrictor in the microchannel. In certain examples,the system can be configured to receive the second column receiveseffluent from the first column when the switching valve is in a firstposition and the third column receives effluent from the first columnwhen the switching valve is in a second position. In some examples, thesystem can include a third detector fluidically coupled to themicrofluidic device through a fourth column between the microfluidicdevice and the third detector. In some embodiments, the system caninclude a vent fluidically coupled to the microfluidic device through arestrictor between the vent and the microfluidic device. In certainembodiments, the system further comprises a vent fluidically coupled tothe microfluidic device through a restrictor between the vent and themicrofluidic device. In certain examples, the system includes a chargingchamber in a microchannel of the microfluidic device.

In an additional aspect, a chromatography system comprising an injector,a column fluidically coupled to the injector, a microfluidic devicefluidically coupled to the column and fluidically coupled to a switchingvalve, the microfluidic device comprising an inlet port to receiveeffluent from the column, an modulating gas port to receive a modulatinggas, and at least one outlet port, each of the ports in fluidcommunication through an internal microchannel in the microfluidicdevice, a restrictor fluidically coupled to a first outlet port of themicrofluidic device and a first detector fluidically coupled to therestrictor is provided.

In certain embodiments, the system comprises a bypass restrictor in theinternal microchannel, the bypass restrictor configured to reduce fluidflow through the microfluidic device. In certain examples, the systemincludes a charging chamber in the microchannel. In other examples, thesystem includes a second restrictor fluidically coupled to a secondoutlet port of the microfluidic device. In some embodiments, the systemcan include a second detector fluidically coupled to the secondrestrictor. In other embodiments, the system can include a thirdrestrictor fluidically coupled to a third outlet port of themicrofluidic device. In certain embodiments, the system can include athird detector fluidically coupled to the third restrictor. In someexamples, the system can include a fourth restrictor fluidically coupledto a fourth outlet port of the microfluidic device. In some embodiments,the system further comprises a fourth detector fluidically coupled tothe fourth restrictor. In certain examples, the system can include asniffer port fluidically coupled to a second outlet port of themicrofluidic device through a restrictor between the second outlet portof the microfluidic device and the sniffer port.

In another aspect, a chromatography system comprising an injector, afirst column fluidically coupled to the injector, a microfluidic devicefluidically coupled to the column and fluidically coupled to a switchingvalve, the microfluidic device comprising an inlet port to receiveeffluent from the column, an modulating gas port to receive a modulatinggas, and at least one outlet port, each of the ports in fluidcommunication through an internal microchannel in the microfluidicdevice, a second column fluidically coupled to a first outlet port ofthe microfluidic device and a first detector fluidically coupled to thesecond column is disclosed.

In certain embodiments, the system can include a bypass restrictor inthe internal microchannel, the bypass restrictor configured to reducefluid flow through the microfluidic device. In other embodiments, thesystem can include a charging chamber in the microchannel. In someexamples, the system can include a restrictor fluidically coupled to asecond outlet port of the microfluidic device. In additional examples,the system can include a second detector fluidically coupled to therestrictor. In certain embodiments, the system can include a thirdcolumn fluidically coupled to a third outlet port of the microfluidicdevice. In some examples, the system can include a third detectorfluidically coupled to the third column. In other examples, the systemcan include a fourth column fluidically coupled to a fourth outlet portof the microfluidic device. In some examples, the system can include afourth detector fluidically coupled to the fourth column. In additionalembodiments, the system can include a sniffer port fluidically coupledto a second outlet port of the microfluidic device through a restrictorbetween the second outlet port of the microfluidic device and thesniffer port.

In an additional aspect, a chromatography system comprising an injector,a microfluidic device fluidically coupled to the injector andfluidically coupled to a switching valve, the microfluidic devicecomprising an inlet port to receive sample from the injector, amodulating gas port to receive a modulating gas, and at least one outletport, each of the ports in fluid communication through an internalmicrochannel in the microfluidic device, a first column fluidicallycoupled to a first outlet port of the microfluidic device, and a firstdetector fluidically coupled to the first column is disclosed.

In certain embodiments, the system can include a bypass restrictor inthe internal microchannel, the bypass restrictor configured to reducefluid flow through the microfluidic device. In other embodiments, thesystem can include a charging chamber in the microchannel. In additionalembodiments, the system can include a restrictor fluidically coupled toa second outlet port of the microfluidic device. In some examples, thesystem can include a second detector fluidically coupled to therestrictor. In other examples, the system can include a third columnfluidically coupled to a third outlet port of the microfluidic device.In additional examples, the system can include a third detectorfluidically coupled to the third column. In other examples, the systemcan include a fourth column fluidically coupled to a fourth outlet portof the microfluidic device. In some examples, the system can include afourth detector fluidically coupled to the fourth column. In otherexamples, the system can include a sniffer port fluidically coupled to asecond outlet port of the microfluidic device through a restrictorbetween the second outlet port of the microfluidic device and thesniffer port.

In another aspect, a chromatography system comprising a first injectorfluidically coupled to a first column, a second injector fluidicallycoupled to a second column, a microfluidic device fluidically coupled tothe first column and the second column, the microfluidic devicecomprising an internal microchannel fluidically coupled to a switchingvalve, a first detector fluidically coupled to the microfluidic device,and a second detector fluidically coupled to the microfluidic device, inwhich the microfluidic device is configured, in a first position, toprovide effluent from the first column to the first detector andeffluent from the second column to the second detector and isconfigured, in a second position, to provide the effluent from the firstcolumn to the second detector and the effluent from the second column tothe first detector in a second position is provided.

In certain embodiments, the system can include a first restrictorbetween the first detector and the microfluidic device and a secondrestrictor between the second detector and the microfluidic device. Insome embodiments, the system can include an internal crossover channelin the microchannel of the microfluidic device, the switching valveconfigured to permit fluid flow through the crossover channel in thesecond position. In some embodiments, at least one of the first andsecond detectors is a mass spectrometer. In other examples, the systemcan include a third column fluidically coupled to the second detectorand between the second detector and the microfluidic device. In certainexamples, the system includes a charging chamber in the microchannel.

In an additional aspect, a chromatography system comprising a firstinjector fluidically coupled to a first column, a second injectorfluidically coupled to a second column, a microfluidic devicefluidically coupled to the first column and the second column, themicrofluidic device comprising an internal microchannel fluidicallycoupled to a switching valve, a first detector fluidically coupled tothe microfluidic device, and a vent fluidically coupled to themicrofluidic device, in which the microfluidic device is configured toprovide effluent from the first column to the first detector andeffluent from the second column to the vent in a first position and toprovide the effluent from the first column to the vent and the effluentfrom the second column to the first detector in a second position isdisclosed.

In certain embodiments, the system can include a first restrictorbetween the first detector and the microfluidic device and a secondrestrictor between the vent and the microfluidic device. In otherembodiments, the system can include an internal crossover channel in themicrochannel of the microfluidic device, the switching valve configuredto permit fluid flow through the crossover channel in the secondposition. In additional embodiments, at least one of the first andsecond detectors is a mass spectrometer. In some embodiments, the systemcan include a third column fluidically coupled to the first detector andbetween the first detector and the microfluidic device. In certainexamples, the system includes a charging chamber in the microchannel.

In another aspect, a chromatography system comprising a first injectorfluidically coupled to a first column, a first microfluidic devicefluidically coupled to the first column and comprising an internalmicrochannel fluidically coupled to a switching valve, a secondmicrofluidic device fluidically coupled to the first microfluidicdevice, the second microfluidic device comprising an internalmicrochannel fluidically coupled to a switching valve and a detectorfluidically coupled to the second microfluidic device is provided.

In certain embodiments, the system can include a second column betweenthe first and second microfluidic devices and fluidically coupled toeach of the first and second microfluidic devices. In other embodiments,the system can include a second detector fluidically coupled to thefirst microfluidic device. In some examples, the system can include asecond detector fluidically coupled to the second microfluidic device.In additional examples, the system can include a second column betweenthe second microfluidic device and the detector and fluidically coupledto each of the second microfluidic device and the detector. In certainexamples, the system includes a charging chamber in a microchannel ofone or more of the first and second microfluidic devices.

In an additional aspect, a chromatography system comprising a firstinjector fluidically coupled to a first column, a second injectorfluidically coupled to a second column, a microfluidic devicefluidically coupled to the first column through a first port andfluidically coupled to the second column through a second port, themicrofluidic device comprising a first charging chamber fluidicallycoupled to the first port and a second charging chamber fluidicallycoupled to the second port, and a switching valve fluidically coupled tothe microfluidic device and to a modulating gas source, the switchingvalve configured, in a first position, to permit flow of a modulatinggas from the modulating gas source to sweep effluent from the firstcharging chamber to a detector fluidically coupled to the microfluidicdevice, the switching valve further configured, in a second position, topermit flow of the modulating gas from the modulating gas source to thesecond charging chamber to sweep effluent from the second chargingchamber to the detector fluidically coupled to the microfluidic device.

In certain embodiments, the system can be configured with a firstdetector fluidically coupled to the first charging chamber and a seconddetector fluidically coupled to the second charging chamber. In otherexamples, the switching valve can be configured to be actuated betweenthe first and second positions to provide pulsed flow from the firstcharging chamber to the detector and from the second charging chamber tothe detector. In additional examples, the switching valve is a 3-waysolenoid valve. In other examples, the detector is a mass spectrometer.

In another aspect, a microfluidic device comprising a laminatedsubstrate comprising a plurality of ports each connected to the other inseries through an internal microfluidic channel is provided. In someexamples, the microchannels can be configured in a desired manner, e.g.,with an internal bypass restrictor.

In certain embodiments, the microfluidic device comprises a switchingvalve fluidically coupled to at least one of the plurality of ports. Inadditional examples, the switching valve is a solenoid valve. In someexamples, the device can include at least one chromatography columnfluidically coupled to a first port of the microfluidic device. In someexamples, a detector can be fluidically coupled to a second port of themicrofluidic device. In certain examples, the detector is a massspectrometer. In other examples, the device may further comprise anadditional chromatography column fluidically coupled to a third port ofthe microfluidic device. In some examples, the device can include arestrictor fluidically coupled to the microfluidic device and positionedbetween the at least one detector and the microfluidic device. Inadditional examples, the device can include a second detectorfluidically coupled to a fourth port of the microfluidic device. In someembodiments, the device can include an additional restrictor fluidicallycoupled to the microfluidic device and positioned between the seconddetector and the microfluidic device. In certain embodiments, the devicecan include a modulating gas source fluidically coupled to a fifth portupstream of the second, third and fourth ports and configured to providethe modulating gas to sweep effluent from the microchannel. In someexamples, the microchannel comprises a charging chamber. In certainexamples, the charging chamber comprises a section having an increaseddiameter as compared to other sections of the microchannel. In someexamples, the modulating gas source is fluidically coupled to chargingchamber and modulating gas can flow from the modulating gas source tothe charging chamber when the switching valve is actuated to a firstposition. In other examples, the modulating gas flow to the chargingchamber is restricted when the switching valve is actuated to a secondposition.

In an additional aspect, a microfluidic device comprising a laminatedsubstrate comprising at least one inlet port and a plurality of outletports each fluidically coupled to the inlet port through a microchannelin the laminated substrate, and an internal bypass restrictor in thelaminated substrate, the internal bypass restrictor fluidically coupledto the microchannel and configured to restrict fluid flow in themicrochannel is provided.

In certain embodiments, the device can include a switching valvefluidically coupled to at least one of the ports. In other embodiments,the switching valve is a solenoid valve. In some examples, the devicecan include at least one chromatography column fluidically coupled to afirst port of the microfluidic device. In additional embodiments, thedevice can include a detector fluidically coupled to a second port ofthe microfluidic device. In some examples, the detector is a massspectrometer. In other examples, the device can include an additionalchromatography column fluidically coupled to a third port of themicrofluidic device. In additional examples, the device can include arestrictor fluidically coupled to the microfluidic device and positionedbetween the at least one detector and the microfluidic device. In someexamples, the device can include a second detector fluidically coupledto a fourth port of the microfluidic device. In certain examples, thedevice can include an additional restrictor fluidically coupled to themicrofluidic device and positioned between the second detector and themicrofluidic device. In some examples, the device can include amodulating gas source fluidically coupled to a fifth port upstream ofthe second, third and fourth ports and configured to provide themodulating gas to sweep effluent from the microchannel. In someembodiments, the microchannel comprises a charging chamber. In otherembodiments, the charging chamber comprises a section having anincreased diameter as compared to other sections of the microchannel. Inadditional embodiments, the modulating gas source is fluidically coupledto charging chamber and modulating gas can flow from the modulating gassource to the charging chamber when the switching valve is actuated to afirst position. In some examples, the modulating gas flow to thecharging chamber is restricted when the switching valve is actuated to asecond position.

In another aspect, a method of modulating the flow of fluid in achromatography system, the method comprising actuating a switching valvebetween a first position and a second position, the first positionpermitting fluid flow from a modulating gas source to a first chargingchamber of a microfluidic device to provide column effluent from thefirst charging chamber to a detector fluidically coupled to themicrofluidic device, and the second position permitting fluid flow fromthe modulating gas source to the second charging chamber of themicrofluidic device to provide column effluent from the second chargingchamber to the detector fluidically coupled to the microfluidic deviceis disclosed.

In certain embodiments, the switching valve is a 3-way solenoid valvethat is actuated at a frequency of about 10 Hz to about 100 Hz. In someembodiments, the method can include balancing pressure in the system byconfiguring the system with a restrictor between the detector and themicrofluidic device. In other examples, the method can include providingthe column effluent from the first charging chamber to a first detectorfluidically coupled to the microfluidic device and providing the columneffluent from the second charging chamber to a second detectorfluidically coupled to the microfluidic device. In some examples, themethod can include balancing the pressure in the system by configuringthe system with a first restrictor between the first detector and themicrofluidic device and with a second restrictor between the seconddetector and the microfluidic device. In certain examples, the methodcan include configuring each of the first and second charging chambersas internal chambers within the microfluidic device. In certainembodiments, the method can include configuring a rate of fluid flowfrom the modulating gas source to be at least five times greater, e.g.,at least ten times greater, than a rate of fluid flow of the columneffluent into the first and second charging chambers.

In another aspect, a system comprising a microfluidic device comprisingan internal microchannel comprising a first charging chamber and asecond charging chamber, the first charging chamber and the secondcharging chamber each fluidically coupled to an inlet port and an outletport of the microfluidic device, and a switching valve fluidicallycoupled to the internal microchannel of the microfluidic device andconfigured to permit flow of a modulating gas through the inlet port andto the first charging chamber in a first position to provide columneffluent from the first charging chamber to the outlet port of themicrofluidic device and to permit flow of a modulating gas through theinlet port and to the second charging chamber in the second position toprovide column effluent from the second charging chamber to the outletport of the microfluidic device. In some examples, where flow of themodulating gas is permitted to the first chamber, the flow of themodulating gas is restricted to the second chamber (or substantially noflow of modulating gas occurs at all to the second chamber). In certainexamples, where flow of the modulating gas is permitted to the secondchamber, the flow of the modulating gas is restricted to the firstchamber (or substantially no flow of modulating gas occurs at all to thefirst chamber).

In certain embodiments, the system can include a detector fluidicallycoupled to the outlet port of the microfluidic device. In someembodiments, the detector is a mass spectrometer. In additionalembodiments, the switching valve is a 3-way solenoid valve. In certainexamples, the system can include a controller electrically coupled tothe mass spectrometer and the switching valve and configured tosynchronize detector readings of the mass spectrometer with modulationof the 3-way solenoid valve. In other embodiments, the system caninclude an injector fluidically coupled to the microfluidic device. Insome examples, the microfluidic device can be configured as a laminatedwafer.

In an additional aspect, a method comprising selecting a fluid flow rateto be used in a chromatography system, and determining an internaldiameter of a column in a chromatography system to provide the selectedfluid is provided. In certain examples, the internal diameter of thecolumn is calculated using the following equation

$d_{c} = \sqrt[4]{\frac{256 \cdot L \cdot p_{a} \cdot \eta}{b \cdot \pi}}$where d_(c) is the internal diameter of the column, L is column length,p_(a) is absolute pressure, η is viscosity of a carrier gas and b is aconstant.

In another aspect, a method comprising selecting a fluid flow rate to beused in a chromatography system, and determining a dimension of arestrictor in a chromatography system to provide the selected fluid flowrate is disclosed. In certain examples, the dimension is an internaldiameter of the restrictor and the internal diameter is calculated usingthe following equation

$d_{r} = \sqrt[4]{\frac{256 \cdot L \cdot p_{a} \cdot \eta}{b \cdot \pi}}$where d_(r) is the internal diameter of the restrictor, L is therestrictor length, p_(a) is absolute pressure, η is viscosity of acarrier gas and b is a constant. In certain examples, the dimension isrestrictor length and the restrictor length is calculated using thefollowing equation

$L_{r} = {\frac{\left\lbrack {{\frac{\pi \times T_{a} \times d_{r}^{4}}{256 \times F_{a} \times p_{a}} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)} - {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}} \right\rbrack}{T_{r\; 1} \times \eta_{r\; 1}} + L_{r\; 2}}$where L_(r1) is the length of the restrictor inside the oven, L_(r2) isthe length of the restrictor inside the detector, η_(r1) is theviscosity of carrier gas at the oven temperature, η_(r2) is theviscosity of carrier gas at the detector temperature, T_(r1) is theabsolute temperature of the oven, T_(r2) is the absolute temperature ofthe detector, p_(i) is the inlet pressure, p_(or) is the outlet pressureand F_(a) is the flow rate.

In another aspect, a method of facilitating use of a chromatographysystem, the method comprising providing a microfluidic device comprisinga laminated substrate comprising a plurality of ports each connected tothe other in series through an internal microfluidic channel isdisclosed.

In an additional aspect, a method of facilitating use of achromatography system, the method comprising providing a microfluidicdevice comprising a laminated substrate comprising at least one inletport and a plurality of outlet ports each fluidically coupled to theinlet port through a microchannel in the laminated substrate, and aninternal bypass restrictor in the laminated substrate, the internalbypass restrictor fluidically coupled to the microchannel and configuredto restrict fluid flow in the microchannel is provided. In certainembodiments, the plurality of the outlet ports can be arranged inseries.

Additional features, aspects, examples and embodiments are described inmore detail below.

BRIEF DESCRIPTION OF THE FIGURES

Certain illustrative embodiments are described in detail below withreference to the accompanying figures in which:

FIGS. 1A and 1B are schematics used to describe the general principlesof operation of a microfluidic device, in accordance with certainexamples;

FIG. 2 is an illustration of a chromatography system having a midpointunion, in accordance with certain examples;

FIGS. 3A-3C show a user interface that can be used in a chromatographysystem to implement flow control, in accordance with certain examples;

FIGS. 4A-4E are diagrams showing fluid flow in the system under variousconditions, in accordance with certain examples;

FIG. 5 is a chromatography system that includes a pneumatic controller,in accordance with certain examples;

FIG. 6 is a chromatography system that includes a switching valve andtwo detectors, in accordance with certain examples;

FIG. 7 is a chromatography system that includes two detectors and asplitter, in accordance with certain examples;

FIG. 8A is graph showing the change in flow rate as a function of theerror in column internal diameter, in accordance with certain examples;

FIG. 8B is a graph showing the change in flow rate as a function of theerror in column length, in accordance with certain examples;

FIGS. 9A and 9B are illustrations of chromatography systems including amicrofluidic device and a switching valve, in accordance with certainexamples;

FIG. 10 is an illustration of a chromatography system including threedetectors, in accordance with certain examples;

FIG. 11 is a cross-section of a microfluidic device showing the internalmicrochannel, in accordance with certain examples;

FIG. 12 is a cross-section of a microfluidic device including fouroutlet ports, in accordance with certain examples;

FIG. 13 is a cross-section of a microfluidic device including anelongated portion in the microchannel, in accordance with certainexamples;

FIGS. 14A and 14B are cross-section of a microfluidic device includingtwo outlet ports and three outlet ports, respectively, each arranged inseries with the other outlet ports, in accordance with certain examples;

FIGS. 15A and 15B show other illustration of microfluidic devices, inaccordance with certain examples;

FIG. 16 is a schematic of a chromatography system that includes a singledetector fluidically coupled to a microfluidic device, in accordancewith certain examples;

FIG. 17 is a schematic of a chromatography system that includes twodetectors each fluidically coupled to a microfluidic device, inaccordance with certain examples;

FIG. 18 is a schematic of a chromatography system that includes threedetectors each fluidically coupled to a microfluidic device, inaccordance with certain examples;

FIG. 19 is a schematic of a chromatography system that includes fourdetectors each fluidically coupled to a microfluidic device, inaccordance with certain examples;

FIG. 20 is a schematic of a chromatography system that includes a singledetector and a sniffer port each fluidically coupled to a microfluidicdevice, in accordance with certain examples;

FIG. 21 is a schematic of a chromatography system that includes twodetectors each fluidically coupled to a microfluidic device and wherebackflushing of a first column can be performed, in accordance withcertain examples;

FIG. 22 is a schematic of a chromatography system that includes twocolumns and a single detector fluidically coupled to a microfluidicdevice, in accordance with certain examples;

FIG. 23 is a schematic of a chromatography system that includes twocolumns and two detectors each fluidically coupled to a microfluidicdevice, in accordance with certain examples;

FIG. 24 is a schematic of a chromatography system that includes threecolumns and two detectors each fluidically coupled to a microfluidicdevice, in accordance with certain examples;

FIG. 25 is a schematic of a chromatography system that includes twodetectors each fluidically coupled to a microfluidic device through acolumn, in accordance with certain examples;

FIGS. 26A and 26B are illustrations of a microfluidic device thatincludes a microchannel where all the ports are arranged in series, inaccordance with certain examples;

FIG. 27 is a photograph showing a microfluidic device and two plates tohold the microfluidic device, in accordance with certain examples;

FIG. 28 is a cross-section of a microfluidic device showing an internalbypass restrictor, in accordance with certain examples;

FIG. 29 is a schematic of a chromatography system that includes twocolumns and two detectors each fluidically coupled to a first and asecond microfluidic device, in accordance with certain examples;

FIGS. 30A and 30B are schematics of a chromatography system with amicrofluidic device that can provide for crossover flow, in accordancewith certain examples;

FIG. 31 is a schematic of another chromatography system with amicrofluidic device that can provide for crossover flow, in accordancewith certain examples;

FIGS. 32A-32D are cross-section sections showing the various layers ofthe microfluidic device, in accordance with certain examples;

FIGS. 33A and 33B show a controller that can be used to actuate aswitching valve, in accordance with certain examples;

FIG. 34 is a graph showing the results of a single peak that has beenmodulated, in accordance with certain examples;

FIGS. 35A-35C show traces of samples from two different columns (FIGS.35A and 35B) and modulation of those sample peaks (FIG. 35C), inaccordance with certain examples;

FIGS. 36 and 37 are schematics of systems that can be used to performsimultaneous analysis of two chromatograms, in accordance with certainexamples;

FIGS. 38 and 39 are schematics of systems configured formultidimensional separations, multiplexed chromatography or multiplexeddetection, in accordance with certain examples;

FIG. 40 is a cross-section of a microfluidic device that includes afirst charging chamber and a second charging chamber, in accordance withcertain examples;

FIG. 41 shows a conventional chromatograph and FIG. 42 shows theprophetic results using modulation and a charging chamber, in accordancewith certain examples;

FIGS. 43A and 43B show flow of fluid through a charging chamber using a2-way switching valve, in accordance with certain examples;

FIGS. 44A and 44B show flow of fluid through a charging chamber using a3-way switching valve, in accordance with certain examples;

FIGS. 45A and 45B show flow of fluid through a first and second chargingchamber using a 3-way switching valve, in accordance with certainexamples;

FIG. 46 shows a microfluidic device that includes an enlargedmicrochannel portion in accordance with certain examples;

FIG. 47 shows a microfluidic device that includes a microchannel havingrestrictions therein, in accordance with certain examples;

FIGS. 48-54 shows illustrations of chromatography systems that can beused, for example, peak splitting, in accordance with certain examples;

FIG. 55 is a photograph showing the diameter of tubing, in accordancewith certain examples;

FIG. 56 is a graph showing the results of flow rate measurements usingthe flow control algorithms described herein and using pressure control,in accordance with certain examples; and

FIG. 57 is schematic showing a microfluidic device that includes aninternal bypass restrictor, in accordance with certain examples.

It will be understood by the person of ordinary skill in the art, giventhe benefit of this disclosure, that the exact size and arrangement ofthe various components shown in the figures can be altered, e.g.,enlarged, stretched, reduced, rearranged or otherwise configureddifferently to provide a desired result or a desired mode of operation.In addition, the particular placement of one component as “upstream” or“downstream” relative to another component may also be altered dependingon the desired results or desired methods to be performed using thetechnology described herein. Unless otherwise noted, fluid flow, e.g.,gas flow, is intended to occur generally from left to right in thefigures, though other flow directions are possible depending on theexact configuration and pressures used, as described in more detailherein. Where possible arrows may be used in certain instances to showthe general direction of fluid flow.

DETAILED DESCRIPTION

The following description is intended to demonstrate some of the useful,novel and non-obvious subject matter provided by the technologydescribed herein. Such description is not intended to be limiting butrather illustrative of the many configurations, embodiments and uses ofthe chromatography systems described herein and the components and usesthereof. The exact shape, size and other dimensions of the componentsshown in the figures can vary depending on the intended use of thedevice, the desired form factor and other factors that will be selectedby the person of ordinary skill in the art, given the benefit of thisdisclosure.

In certain embodiments, the devices, methods and systems describedherein can be used in fluid chromatography systems. Fluid chromatographysystems are intended to include, but not be limited to, gaschromatography systems, liquid chromatography (LC) systems,supercritical fluid (SCF) chromatography systems and combinations ofthese illustrative fluid chromatography systems. Certain specificexamples are described below with particular reference to gaschromatography (GC) systems, but similar principles and configurationsmay be used with fluid chromatography systems other than GC systems.

In the systems disclosed herein and illustrated in the figures, thegeneral term “detector” is often used. The detector may be any commonlyused GC, LC or SCF detector including, but not limited to, a flameionization detector (FID), a flame photometric detector (FPD), a thermalconductivity detector (TCD), a thermionic detector (TID), anelectron-capture detector (ECD), an atomic emission detector (AED), aphotoionization detector (PI), an electrochemical detector, afluorescence detector, a UV/Visible detector, an infrared detector, anuclear magnetic resonance detector or other detectors commonly usedwith GC, LC or SCF. In addition, the detector may be a massspectrometer, an external detector such as, for example, a dischargeionization detector (DID) or a sulfur chemiluminescence detector (SCD)or other suitable detectors and devices that can be hyphenated to a gaschromatography device or other fluid chromatography devices, e.g., thoseusing capillary columns.

In certain embodiments, the terms “microfluidic device,” “switch,” or“microfluidic device” are used interchangeably herein. Microfluidicdevices are described in many different instances and are typicallyconfigured to provide fluid flow from at least one inlet port to one ormore outlet ports. The microfluidic devices may also be configured toprovide fluid flow to two or more devices that can be fluidicallycoupled to outlet ports of the microfluidic device. The microfluidicdevice can take many different forms such as, for example, a laminatedwafer including a plurality of layers that when assembled provide one ormore internal microfluidic channels. “Fluidically coupled” is usedherein to refer to the case where fluid can flow between two or morecomponents. Fluid flow can be permitted between the components by, forexample, switching or opening a valve between the components, whereasfluid flow can be restricted between the components, for example, byswitching or closing the valve. Where two or more components arefluidically coupled, fluid is not necessarily flowing between them atall times. Instead, depending on the other components of the system andtheir operational state, fluid can flow between the two fluidicallycoupled components under certain configurations and arrangements. In thecase of a switching valve positioned between two components, forexample, the two components can remain fluidically coupled when thevalve is in the closed position even though no fluid is flowing betweenthe components.

In certain embodiments, the flow control algorithms and methodsdescribed herein are applicable to restrictors, columns, transfer linesor other tubing such as, for example, capillary tubing. For example, thediameters and lengths of the restrictors, columns, transfer lines, etc.can be determined using the algorithms, and the description providedherein that is directed to a particular device, e.g., a restrictor, maybe applied to a different device, e.g., a column, of the system.

In certain examples, the components described herein can be connected toeach other through tubing, fittings, ferrules or other devices that canprovide for substantially fluid tight seals and can provide a fluid flowpath between two or more selected components. The lengths, diameters andother parameters for such additional components can be determined basedon experimentation or using the length and diameter calculationsdescribed herein.

In certain examples, the devices and system described herein can be usedin many different types of chromatography systems. It is desirable thatthe devices be configured for use in either heartcut or solvent dumpsystems. In heartcut systems, selected species or peaks in the samplemay be sent to two or more different columns or detectors. Heartcutsystems may be particularly advantageous where poor resolution of twopeaks is achieved. Those peaks can be sent to a different column havinga different separation media or mechanism. For example, a firstconventional column of 30 meters length with an internal diameter of0.25 or 0.32 mm can be used to provide a first separation stage. Aselected portion of the column effluent can then be passed to a secondcolumn having a different stationary phase, length, internal diameter orother characteristics that can be used to separate components in thatportion of the first column effluent. In a solvent dump system, theamount of solvent sent to a detector may be reduced. For example, it maybe desirable to reduce the solvent volume sent to a detector such as amass spectrometer. A crude separation can first be performed on a firstcolumn, e.g., a large internal diameter, low resolution column. Only thecomponents of interest can be sent to a second column, which can be ahigher resolution column. To account for difference in pressure, one ormore restrictors may be used in the system. For example, there is littlepressure differential across a large internal diameter column, andpressures needed to direct the reverse flow across an orifice can causea large reduction in the flow through the first column. To reduce thiseffect, a restrictor can be used to increase the overall pressure in thesystem. Use of restrictors and their effects on pressure are describedin more detail herein.

In certain examples, the devices, systems and methods described hereincan include a microfluidic device. The microfluidic device can beconfigured to split flow from a column, to switch the flow between twoor more outlet ports or to provide fluid flow to other ports or in otherdirections. Certain specific configurations of a microfluidic device aredescribed below. These configurations are merely illustrative and othersuitable configurations are possible. In certain embodiments, themicrofluidic devices described herein are operative to direct gas flowusing differential pressures from external gas supplies or pressureregulators. These differential pressures can be used to change thedirection of gas flow eluting from a chromatography column between twoor more outlet ports. Such operation can have advantages overtraditional mechanical-based valve systems including, for example, inputand output flow rates are undisturbed resulting in no or littlealteration of retention times, the devices can be fabricated from lowthermal mass components to avoid or reduce the likelihood of cold spots,there are no moving parts (or few moving parts where one or more valvesare present), the internal volumes of the channels in the switch can beminimal to reduce peak dispersion and adsorption effect, the responsetime is very fast allowing narrow cuts to be switched between outputswhich permits it use with modern capillary columns, and internalsurfaces can be generally inert and/or deactivated to enable use withlabile analytes. Other advantages are also possible depending on theexact configuration of the system.

In accordance with certain examples, the flow rate control describedherein may be used by itself or in combination with one or moremicrofluidic devices. For example, a microfluidic device may beconfigured as a heartcutting accessory or module that includes one ormore microchannels. In other configurations, the microfluidic device canbe configured to split effluent from a column between multipledetectors. Other configurations of a microfluidic device will berecognized by the person of ordinary skill in the art, and certainillustrative configurations are described herein.

In certain embodiments, the devices described herein can be used toprovide blends of fluids, e.g., gas or liquid blends, that can be usedin chromatographic separations or can be used, for example to study gasphase reaction kinetics. For example, two or more different gases can beprovided in desired amounts using the flow control algorithms describedherein. The gases can be mixed in a microfluidic device (or otherdevice). For example, a first gas can be introduced into a first port ofa microfluidic device and a second gas can be introduced into a secondport of the microfluidic device. The gases can be mixed, e.g., using aninternal buffer, charging chamber or other desired internal channel, andoutputted to a reaction chamber, detector or other suitable device. Itwill be within the ability of the person of ordinary skill in the art,given the benefit of this disclosure, to use the microfluidic devicesdescribed herein for these and other uses.

Pressure balanced systems were pioneered by Dr. David Deans of ICIChemicals in the late 1950s and remain a popular choice in severalimportant GC applications. For example, the PreVent, Protect, MS Ventand Ozone Precursor systems commercially available from PerkinElmer(Waltham, Mass.) all utilize this technique. These techniques aresufficiently powerful that, in many instances, there is no other way ofperforming a particular analysis or their use makes significantimprovements with respect to throughput or quality of the results.

With existing pressure balance systems, there are some drawbacks. It isnot possible to directly or explicitly control the flow rate of carriergas through the column. Many users prefer to specify the flow raterather than the inlet pressure for carrier gas control through thecolumn. In most instances, this gives more consistent chromatographicperformance. It is also not feasible to control the flow rate of carriergas into the detector. The response of most GC detectors is highlysensitive to gas flow rate and so users would prefer to use flow controlto minimize baseline drift and provide consistent analyte response. Theapplied carrier gas pressures can also be very difficult to set up andrequires a substantial amount of understanding on the part of the user.Certain embodiments described herein can permit control of the flow ratethrough a column by controlling or specifying the carrier gas flow ratethrough a column.

To facilitate a better understanding of the microfluidic devicesdescribed herein, a generalized operation principle of a microfluidicdevice is described in reference to FIGS. 1A and 1B. Referring to FIG.1A, effluent from a column enters a T-shaped piece at a site or point 52in the direction of an arrow 50. A switching valve 65, e.g., a solenoidvalve, MEMS device or other suitable devices, can be switched to a firstposition or modulated open to at least some degree to permit carrier gasfrom the gas source 67 to flow into the T-shaped piece at point 54. Thegas pressure provided by the source 67 is at a slightly higher pressurethan the gas pressure at point 52. The pressure at point 54 is slightlyhigher than at points 52 and 56 such that carrier gas will flow frompoint 54 towards points 52 and 56 and push or direct effluent from thecolumn toward point 56. The effluent will exit the T-shaped piece atport 58 in a direction as shown by an arrow 57 in FIG. 1A. In addition,carrier gas with substantially no effluent from the column will exit theT-shaped piece at port 60. A needle valve 62 at the center of the deviceis operative to maintain a trickle flow of carrier gas through theunswept gas line so that sample does not diffuse into those areas frompoint 56.

In certain examples, the switching valve 65 can be switched to a secondposition such that gas flow in the T-shaped piece is altered orreversed. Referring to FIG. 1B, the switching valve 65 is actuated suchthat gas flow from the pressure source 67 to the point 56 such thatpressure at the point 56 is higher than at points 52 and 54. Theeffluent from the column will exit the T-shaped piece at port 60 in adirection as shown by an arrow 59 in FIG. 1B. In addition, carrier gaswith substantially no effluent from the column will exit the T-shapedpiece at port 58. The system shown in FIGS. 1A and 1B is designed tooperate when the pressure at points 58 and 60 are substantially thesame, e.g., when the pressures are balanced at these points. Asdescribed in detail below, the microfluidic devices disclosed herein canbe used in such pressure balanced systems to direct the flow of specieseluting from a column to a desired port fluidically coupled to adetector, vent, column or other component.

In certain examples where a microfluidic device includes a switchingvalve, the switching valve may be operative to connect (or disconnect)two or more fluid flow paths such that fluid can flow between the flowpaths when connected and fluid flow is restricted when the flow pathsare disconnected. Illustrative switching valves include, but are notlimited to, a valve such as, for example, a flow control valve, asolenoid valve or a photovac valve, MEMS devices, metal laminatedconstructs with a laminated membrane operative to open and close achannel underneath it, electromechanical valves, pneumatically operatedmembrane valves, motor operated needle valves and other suitable devicesthat can restrict flow in one state and permit flow in another state. Incertain examples, the switching valve can be integrated into themicrofluidic devices disclosed herein, whereas in other examples, theswitching valve may be separate from the microfluidic device. Forexample, where the microfluidic device is placed in an oven, theswitching valve can be placed external to the oven and coupled to themicrofluidic device through suitable supply lines and/or tubing. Suchexternal placement can be particularly desirable where the high oventemperatures can adversely affect performance of the switching valve. Insome examples, the switching valve can be surface mounted to an externalsurface of the oven so that the length of any tubing between theswitching valve and the microfluidic device can be reduced.

In certain embodiments, the microfluidic devices described herein mayinclude, or be configured as, a wafer, a laminate or other suitablyconfigured device that can provide one or more fluid flow paths from aninlet to two or more potential outlets. The device can be configured toprovide flow control of species within a column, detector or otherportions fluidically coupled to the device. For example, themicrofluidic device may be configured with one or more microchannels toprovide for switching or selective flow of gas within a system.Illustrative such systems and devices are described in more detailbelow. Such microfluidic devices can also permit the control of carriergas flow through a separation column to simplify the overall setup anduse of an instrument by an end-user. These and other features andconfigurations are described by way of illustration using gaschromatography systems and reference to certain specific embodiments.

In a typical capillary column setup, gas flows from the injector inother ways than just out into the column itself. These pathways include,but are not limited to, splitters, septum purge and an occasional minorleak. Because of these other pathways, regulating the rate of carriergas flow into the injector does not normally control the actual rate offlow through the column itself. To circumvent this difficulty, most GCsactually control the carrier gas pressure and not explicitly the flowrate. The pressure is applied to deliver the set flow rate according tothe Hagen-Poiseuille relationship shown in Equation 1

$\begin{matrix}{F_{o} = \frac{\pi \times d_{c}^{4} \times \left( {p_{i}^{2} - p_{0}^{2}} \right)}{256 \times L \times \eta \times p_{o}}} & (1)\end{matrix}$where F_(o) is the flow rate at the outlet, d_(c) is the internaldiameter of the column, L is the column length, η is the viscosity ofthe carrier gas at the set temperature, p_(i) is the gas pressure at theinlet and p_(o) is the gas pressure at the outlet. Using the aboveequation, a user can enter into the user interface the details of thecolumn geometry (d and L), the carrier gas type (to allow the viscosityto be calculated correctly) and the column outlet pressure(p_(o)—normally set to ambient pressure or vacuum, for MS systems). TheGC system will have knowledge of the column temperature (to enable theviscosity to be calculated) and so it can calculate the inlet pressure(p_(i)) needed to deliver a required flow rate.

Once the system is set up and running, the only potential variable isthe gas viscosity which changes if the column temperature is increasedduring an oven temperature program. Using Equation 1, the system canadjust the inlet pressure, p_(i), to maintain the set carrier gas flowrate. While this approach has been widely adopted for carrier gas flowrate control in many successful GC designs, it is not entirely accuratefor use in pressure balanced systems and so an alternative approach tocarrier gas control is desirable.

In certain examples, a typical pressure balanced system is shown in FIG.2 such as, for example, a system configured as a single column backflushconfiguration or a heartcut configuration. The system 100 includes aninjector 110 that can provide a split flow in direction 115. A column120 is fluidically coupled to the injector 110 to receive a carrier gas,from a gas source 105, and any sample that may have been introduced intothe system through the injector 110. A pressure balanced system has atleast two active components through which a gas is flowing, the column120 and a restrictor 130.

The flow rate through the column 120 is generally a function of itsinlet pressure at the injector (p₁) and its outlet pressure at themidpoint pressure (p₂) at a midpoint union 125, whereas the flow ratethrough the restrictor 130 into the detector 135 is controlled by itsinlet pressure at the midpoint p₂ and its outlet pressure at thedetector (p_(o)). These two flow rates are not necessarily the same (infact in most applications, they are desirably different) and may beindependently controlled by varying combinations of the pressures p₁ andp₂ using independent gas sources 105 and 122.

The flow rates of carrier gas through the column 120 and the restrictor130 may each still be calculated using Equation 1—they just havediffering inlet and outlet pressures. To provide carrier gas flowcontrol within just the column 120, the pressure at the midpoint as theexit pressure can be used.

In certain examples and referring to FIG. 3A, a graphical user interfacescreen 310 of a Clarus GC is shown. Currently, the column exit pressurecan only be set to ambient (implied if vacuum not selected) or vacuum(for MS, for example). To enable flow control of the column when fittedto a pressure balance system, it would be desirable for the user to havethe ability to enter the outlet pressure, for example, as shown inscreen 320 in FIG. 3B or in screen 330 in FIG. 3C. Such modificationpermits a user to explicitly control the flow rate through a GC columnin a pressure balanced system to provide a constant gas flow through acolumn during temperature programmed chromatography.

In certain examples, flow control through both a column and a restrictormay be performed using the devices and methods described herein. Tocontrol the flow through the restrictor, its dimensions and its outletpressure (ambient or vacuum) should be known or measurable. Theremainder of the information will be the same as that for the column.The flow rate can be controlled by setting the restrictor inlet pressure(e.g., the midpoint pressure) according to Equation 1. In some examples,a PPC pressure module such as, for example, those used in a PerkinElmerPreVent system can be used as a carrier supply (with flow rate controlalgorithms) rather than just a passive pressure regulator. Once themidpoint PPC module is configured, the injector pressure, e.g., thecolumn inlet pressure, would be set to deliver the set flow rate usingthe PPC midpoint pressure setting for the outlet pressure in Equation 1.The whole process can automatically track an oven temperature program ifthe column outlet pressure is to be dynamically linked to the midpointpressure as shown in FIG. 3C. This approach can provide independentcontrol of the gas flows through both the column and the restrictor toprovide constant gas flow through the column during temperatureprogrammed chromatography and constant gas flow into the detector.Independent and explicit control of the two gas flows also provides fora more user friendly setup and operation of a pressure balanced system.

In accordance with certain examples, to consider some of theimprovements flow control can provide, a configuration for pressurebalancing is shown in FIG. 4A. Adjustment of the midpoint pressureaffects the respective flow rates in the column and restrictor inopposite ways. An increase in the midpoint pressure reduces the flowrate through the column but increases the flow rate through therestrictor. Reducing this pressure has an opposite effect on both flows.Referring to FIG. 4A, a pressure balancing system 400 is shown whichincludes an injector 410 that has a split flow at point 415, a column420 fluidically coupled to the injector 410, a midpoint union 425 in aflow path to a restrictor 430, which itself is fluidically coupled to adetector 435. With the midpoint pressure set very low such that p₂ isless than p₁, the flow rate F_(c) through the column will be higher thanthe flow rate F_(t) through the restrictor and so flow of gas will notoccur from the midpoint union 425 as shown in FIG. 4A. Instead, gasflowing from the column 420 will flow up the midpoint supply line 422 ata flow rate of F_(m) causing loss of sample and potential contaminationof the pneumatics system.

In certain examples and referring to FIG. 4B, where the flow ratesthrough the column F_(c) and the restrictor F_(t) are the same, thenthere is substantially no flow to or from the midpoint union 425, e.g.,F_(m) is about zero. Under the situation shown schematically in FIG. 4B,the mid-point pressure is referred to as the natural mid-point pressureand the system can be considered as pressure balanced. This pressurebalancing state can serve as a baseline for the pressure settings.

In certain embodiments and referring to FIG. 4C, where the flow rateF_(c) through the column is less than the flow rate F_(t) through therestrictor, for example, by increasing the flow rate F_(m) at themidpoint union 425, gas will flow through the midpoint regulator 421into the midpoint union 425 and mix with the column effluent going intothe restrictor 430 and the detector 435. As the midpoint pressure isprogressively increased, the flow rate of gas from the column willsteadily decrease until a point is reached where the midpoint pressureis the same as the injector pressure (see FIG. 4D). The flow rate F_(c)through the column will become zero and any chromatography would stop.Under these conditions, gas flow into the detector 435 is still beingmaintained solely by the midpoint regulator 421.

In certain examples and referring to FIG. 4E, if the midpoint pressureis raised above that of the injector such that p₂ is greater than p₁,then the flow of gas F_(c) through the column is reversed and may exitthe system at the split point 415. This situation is not particularlyadvantageous for chromatographic separations, but it may be used forbackflushing. For example, heavy samples or samples that are difficultto elute from the column can be driven from a column with backflushingafter the species of interest have eluted from the column.

In accordance with certain examples, the natural midpoint of the systemcan be advantageously used in the methods and configurations disclosedherein. As discussed herein, the natural midpoint represents thethreshold between losing sample and diluting the midpoint and so itsdetermination can increase the overall accuracy of the methods anddevices described herein. To determine the natural midpoint, a systemsuch as that shown in FIG. 5 can be used. The system 500 includes aninjector 510 fluidically coupled to a column 520 and a carrier gassource 505. A midpoint union 525 is between the column 520 and arestrictor 530. The system 500 also includes a pressure transducer 540,a switching valve 550, and a proportional valve 555 each electricallycoupled to a controller 545. During normal operation, the pressuretransducer 540 can monitor the gas pressure of the gas as it flows inthe system. The internal controller 545 uses this information to adjustthe proportional valve 555 positioned upstream of the pressuretransducer 540. In this way, closed-loop control can maintain thepressure at a set value.

To establish the natural midpoint pressure, the switching valve 550 isactuated so there is no flow into or out of the midpoint union 525(assuming that there are no leaks). The flow rates through the column520 and the restrictor 530 will now be substantially the same. As gasflows through the column 520 and out through the restrictor 530, thepressure at the midpoint will eventually reach a stable value—thenatural midpoint pressure. The flow through the column 520 can becalculated using Equation 1 or estimated from tables. If the flow needsto be adjusted, the inlet pressure p₁ can be changed, the midpointpressure is given time to stabilize and the calculation repeated untilthe desired flow rate is obtained.

Once the correct flow rate has been established and the correspondingnatural midpoint pressure is known, then the switching valve 550 can beactuated to permit flow of gas and the midpoint pressure can be set to 1or 2 psi above the natural midpoint pressure. This slight increase inthe set pressure over the natural pressure provides a positive flow ofgas from the midpoint regulator to prevent sample from diffusing intothe supply line 552. It also serves to maintain the pressure balance asthe oven temperature changes. Setting up a pressure balanced system inthe traditional way using differential pressure control is a longtedious process which tends to put many potential users off or causedifficulties in the setup and subsequent performance.

In certain embodiments, with explicit control of the flow rate in thecolumn and the restrictor, system setup is greatly simplified andoverall accuracy and precision can be increased. The system can beconfigured such that the flow rate through the restrictor is less thanthe flow rate through the column to ensure correct operation. The systemuser needs only to enter the respective flow rates in the analyticalmethod and the differential flow control can preserve the correctbalance between the column and the restrictor and provide a constantflow rate of gas through the column and into the detector. This approachcan be used in the simple situation of single column backflushing asdescribed earlier but also with heartcutting and splitting as shown inFIGS. 6 and 7. Referring to FIG. 6 where a heartcut configuration isshown, the system 600 includes an injector 610 fluidically coupled to apressure regulator 605 through a supply line 607. The injector 610 isfluidically coupled to a column 620 through a supply line 617. Thecolumn 620 is fluidically coupled to a microfluidic device 630 through asupply line 622. The microfluidic device 630 is fluidically coupled to amidpoint pressure regulator 635 through a supply line 637. Detectors 650and 660 are fluidically coupled to the microfluidic device 630 throughrestrictors 640 and 645, respectively. The restrictors 640 and 645 aredesirably matched or substantially the same such that the flow rateF_(r1) through the restrictor 645 is substantially the same as the flowrate F_(r2) through the restrictor 650. Methods of determiningrestrictor lengths and diameters to provide a desired flow rate aredescribed herein.

In certain examples and referring to FIG. 7, a pressure regulatedsplitter system is shown. The system 700 includes an injector 710fluidically coupled to a pressure regulator 705 through a supply line707. The injector 710 is fluidically coupled to a column 720 through asupply line 717. The column 720 is fluidically coupled to a midpointpressure regulator 735 through supply lines 722 and 737 at a union orsplit 727. Detectors 750 and 760 are fluidically coupled to the midpointpressure regulator 735, through the union or split 727, throughresistors 740 and 745, respectively. In operation of the system 700, theflow rate can be set for one of the restrictors 740 and 745, and theother restrictor can be displayed and maintained but not controlledindependently of the other restrictor. The actual flow rate in both thecolumn and the restrictor can be determined using Equation 1 for thecalculations. The length of the column and its diameter may be inputtedby the users, based on the column specifications provided by the columnsupplier. From Equation 1, the column flow rate has a fourth orderdependence on column diameter. Thus, an error in diameter of the columncan lead to a large error as shown prophetically in the graph of FIG.8A. As shown in FIG. 8A, an error of just 2% in the internal columndiameter, e.g., 5 microns on a 250 micron inner diameter column, isenough to cause an inaccuracy of almost 8% in the applied flow rate.With respect to column length, there is a reciprocal relationshipbetween column length between flow and column length as shown in FIG.8B. In this case, an error of 2% in column length, e.g., about a 60centimeter error on a 30 meter column, will produce about a 2% error inthe calculated flow rate. This error can lead, however, to additionalerrors in the flow rate assumptions.

In certain examples, one solution to any potential inaccuracy is toconsider the provision of a geometric factor (GF) that can be applied toa particular column. The GF can be approximated using Equation 2.

$\begin{matrix}{{GF} = \frac{d_{c}^{4}}{L}} & (2)\end{matrix}$

The GF is constant for any given column and should be simple toestablish by simple experiment (by either the supplier or the end user).Because this measurement is empirical, it will apply directly to a givencolumn without making any assumptions about its geometry. Inserting thegeometric factor in Equation 1 provides Equation 3.

$\begin{matrix}{F_{o} = \frac{\pi \times {GF} \times \left( {p_{i}^{2} - p_{0}^{2}} \right)}{256 \times \eta \times p_{o}}} & (3)\end{matrix}$

To calculate the flow rates, the inlet (p_(i)) and outlet (p_(o))pressures and the temperature to calculate the viscosity must be enteredor known. In a typical configuration, these parameters are known by thecontroller or entered by the user.

In accordance with certain examples, the situation with restrictor flowrate control is similar to that of the column. The restrictor isgenerally much shorter than the column and so it is much easier tomeasure its length. The internal diameter is normally much smaller andso small errors in its measurement will have a much greater impact onthis flow rate of gas passing through it. The application of a GF forthe restrictor is therefore just as desirable as it is for the column.One other aspect of the restrictor that can be considered is that partof it (or possibly most of it in the case of an MS) will reside insidethe body of the detector. Thus, different sections of the restrictorwill be at different temperatures and so Equations 1 and 3 may not beentirely accurate. This aspect can be addressed by using an approachtaken to calculate flow rates through a serially connected transfer lineand column for the TurboMatrix thermal desorption systems as given inEquation 4 and as described, for example, in commonly assigned U.S. Pat.Nos. 7,219,532 and 7,468,095, the entire disclosure of each of which ishereby incorporated herein by reference in its entirety.

$\begin{matrix}{F_{o} = {\frac{\pi \times {Tr}}{256 \times p_{o}} \times \frac{\left( {p_{i}^{2} - p_{o}^{2}} \right)}{\left( \frac{T_{c} \times \eta_{c}}{{GF}_{c}} \right) + \left( \frac{T_{r} \times \eta_{r}}{{GF}_{r}} \right)}}} & (4)\end{matrix}$In equation (4), F_(o) is the flow rate at the restrictor outlet (at thetemperature and pressure at that location), GF_(c) is the columngeometric factor, GF_(r) is the restrictor geometric factor, d_(c) isthe column internal diameter and L_(c) is the column length (fordetermining GF_(c)), d_(r) is the transfer line internal diameter andL_(r) is the length of the transfer line (for determining GF_(r)), η_(c)is the viscosity of the carrier gas in the column, η_(r) is theviscosity of the carrier gas within the restrictor, T_(c) is theabsolute temperature of the column, T_(r) is the absolute temperature ofthe transfer line, p_(i) is the absolute pressure of the carrier gas atthe column inlet and p_(o) is the absolute pressure of the carrier gasat the restrictor outlet. Equation 4 can be used in place of Equation 3to provide a more accurate calculation of the flow rate.

In certain examples, the length of a restrictor of a selected internaldiameter can be calculated based on the desired flow rate through acolumn of specified geometry. Such dimensions can depend, at least inpart, on the temperatures and gas pressures desired in the system. Oneconfiguration of a system with a restrictor is shown in FIG. 9A. Thesystem of FIG. 9A includes a split injector 905 fluidically coupled to afirst column 910 and a carrier gas source 902. A microfluidic device 920is fluidically coupled to the first column 910 and is configured todirect species eluting from the first column 910 to a desired component.For example, column effluent can be directed to a first detector 930through a restrictor 925, or column effluent can be directed to a secondcolumn 935 and onto a second detector 940 using the microfluidic device920 and a switching valve 945.

In certain examples, the dimensions and geometry of the restrictor 925can be selected to further balance the pressures in the system. Atypical restrictor includes a piece of deactivated fused silica tubingof known internal diameter that can be cut to a length calculated toprovide substantially the same flow rate of gas the through the columnunder a particular applied pressure and temperature. The length of therestrictor can be determined by trial and error, where the length of therestrictor is progressively shortened until the correct flow rate isachieved. However, this process is cumbersome and can take a substantialamount of time to determine the proper restrictor length. Incrementalshortening of the restrictor may also not take into account thedownstream effects of detector temperature on the flow rates through thecolumn and the restrictor, which can have a significant effect on theactual flow rate to cause a pressure imbalance in the system. Wheremultiple detectors are present and used at different pressures, e.g., anFID (ambient pressure) and a MS detector (vacuum pressure), the pressureimbalancing may be even greater.

In certain embodiments, the restrictor geometry and length can becalculated to match or substantially match the gas flows in a selectedcolumn based on oven and detector temperature and detector operatingpressure. Current calculations assume that the restrictor is of uniformlength and temperature. The flow rate can be calculated according toequation (5)

$\begin{matrix}{F_{a} = \frac{\pi \times d_{r}^{4} \times T_{a} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)}{256 \times L_{r} \times p_{a} \times \eta \times T_{r}}} & (5)\end{matrix}$where F_(a) is the restrictor outlet flow rate at ambient temperatureand pressure, d_(r) is the internal diameter of the restrictor, T_(a) isthe ambient absolute temperature, p_(i) is the carrier gas absolutepressure at the restrictor inlet, p_(or) is the carrier gas absolutepressure at the restrictor outlet, L_(r) is the length of therestrictor, p_(a) is the ambient absolute pressure, η is the viscosityof the carrier gas at the restrictor temperature, and T_(r) is therestrictor absolute temperature.

To determine the restrictor length to match a desired gas flow in acolumn, two simultaneous equations based on equation (5) can be used tosolve for L_(r), which provides equation (6)

$\begin{matrix}{L_{r} = {L_{c} \times \frac{d_{r}^{4}}{d_{c}^{4}}}} & (6)\end{matrix}$where d_(c) is the internal diameter of the column, and L_(c) is thelength of the column. Equation 6 can be used, for example, where thetemperature and the applied inlet and outlet pressures are the samebetween the column and the restrictor.

Where two or more detectors or a detector and a vent or any two devicesoperated at different pressure are present, Equation (5) can be used toobtain Equation (7)

$\begin{matrix}{L_{r} = {L_{c} \times \frac{d_{r}^{4} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)}{d_{c}^{4} \times \left( {p_{i}^{2} - p_{oc}^{2}} \right)}}} & (7)\end{matrix}$where p_(oc) is the carrier gas absolute pressure at the column outlet.

In certain embodiments, to take into account the effect of detectortemperature on the gas flow rates through both the column and therestrictor, the relationship shown in Equation (8a) can be used.

$\begin{matrix}{F_{a} = {\frac{\pi \times T_{a}}{256 \times p_{a\;}} \times \frac{\left( {p_{i}^{2} - p_{o}^{2}} \right)}{\left( \frac{T_{t} \times \eta_{t} \times L_{t}}{d_{t}^{4}} \right) + \left( \frac{T_{c} \times \eta_{c} \times L_{c}}{d_{c}^{4}} \right)}}} & \left( {8a} \right)\end{matrix}$

In Equation (8a), F_(a) is the flow rate at the column outlet, d_(c) isthe column internal diameter, d_(t) is the transfer line (or restrictor)internal diameter, L_(c) is the column length, L_(t) is the transferline (or restrictor) length, η_(c) is the viscosity of the carrier gaswithin the column, η_(t) is the viscosity of the carrier gas within thetransfer line (or restrictor), T_(c) is the absolute temperature of thecolumn, T_(t) is the absolute temperature of the transfer line (orrestrictor), T_(a) is the absolute ambient temperature, p_(i) is theabsolute pressure of the carrier gas at the inlet, p_(o) is the absolutepressure of the carrier gas at the outlet and p_(a) is the absoluteambient pressure. Equation (8a) can be generalized for any number ofserially connected columns or restrictors of differing internaldiameter, length or temperature, as shown in Equation 8(b).

$\begin{matrix}{F_{a} = {\frac{\pi \times T_{a}}{256 \times p_{a}} \times \frac{\left( {p_{i}^{2} - p_{o}^{2}} \right)}{\left( \frac{T_{1} \times \eta_{1} \times L_{1}}{d_{c\; 1}^{4}} \right) + \left( \frac{T_{2} \times \eta_{2} \times L_{2}}{d_{c\; 2}^{4}} \right) + {\ldots\mspace{14mu}\left( \frac{T_{n} \times \eta_{n} \times L_{n}}{d_{cn}^{4}} \right)}}}} & \left( {8b} \right)\end{matrix}$The column and the restrictor of uniform diameter are in a GC oven andare at a different temperature than a detector. Equation (8a) can bemodified for the restrictor and the column to provide Equations (9) and(10) for the restrictor and the column, respectively.

$\begin{matrix}{F_{a} = {\frac{\pi \times T_{a} \times d_{r}^{4}}{256 \times p_{a\;}} \times \frac{\left( {p_{i}^{2} - p_{or}^{2}} \right)}{{T_{r\; 1} \times \eta_{r\; 1} \times L_{r\; 1}} + {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}}}} & (9)\end{matrix}$In Equation (9), L_(r1) is the length of the restrictor inside the oven,L_(r2) is the length of the restrictor inside the detector, η_(r1) isthe viscosity of carrier gas at the oven temperature, η_(r2) is theviscosity of carrier gas at the detector temperature, T_(r1) is theabsolute temperature of the oven and T_(r2) is the absolute temperatureof the detector.

$\begin{matrix}{F_{a} = {\frac{\pi \times T_{a} \times d_{c}^{4}}{256 \times p_{a\;}} \times \frac{\left( {p_{i}^{2} - p_{oc}^{2}} \right)}{{T_{c\; 1} \times \eta_{c\; 1} \times L_{c\; 1}} + {T_{c\; 2} \times \eta_{c\; 2} \times L_{{c\; 2}\;}}}}} & (10)\end{matrix}$In Equation (10), L_(r1) is the length of the restrictor inside theoven, L_(c2) is the length of the restrictor inside the detector, η_(c1)is the viscosity of carrier gas at the oven temperature, η_(c2) is theviscosity of carrier gas at the detector temperature, T_(c1) is theabsolute temperature of the oven and T_(c2) is the absolute temperatureof the detector.

In certain examples, Equation (10) can be used to calculate the pressureto apply to the column to deliver a required flow rate through thecolumn by rearranging it as Equation (11).

$\begin{matrix}{p_{i} = \sqrt{{\frac{F_{a} \times 256 \times p_{a}}{\pi \times T_{a} \times d_{c}^{4}} \times \left( {{T_{c\; 1} \times \eta_{c\; 1} \times L_{c\; 1}} + {T_{c\; 2} \times \eta_{c\; 2} \times L_{c\; 2}}} \right)} - p_{oc}^{2}}} & (11)\end{matrix}$Once the inlet pressure is calculated for the column at a specificgeometry, temperatures and outlet pressure, the length of the restrictormay be calculated using the rearranged form of Equation (9) as shown inEquation (12).

$\begin{matrix}{L_{r} = {\frac{\left\lbrack {{\frac{\pi \times T_{a} \times d_{r}^{4}}{256 \times F_{a} \times p_{a}} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)} - {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}} \right\rbrack}{T_{r\; 1} \times \eta_{r\; 1}} + L_{r\; 2}}} & (12)\end{matrix}$Using Equation (12), the length of a restrictor of known internaldiameter that can provide a desired flow rate to balance the system canbe calculated. In particular, the length of a restrictor of knowninternal diameter to balance the flow rate in another channel takinginto account detector length, temperature and pressure of the otherdetector can be determined. In use, the algorithms can be implemented insoftware such that a user can enter a desired flow rate and therestrictor lengths and diameters to provide such desired flow rate,under specified column parameters and temperatures, can be displayed ina user interface to facilitate use of the system.

Certain embodiments described herein include the use of an additionalcolumn in the chromatography system. The additional column is used inplace of a restrictor and is typically present when heartcutting isdesired. Unlike fused silica restrictors, a user is unlikely to cut offpieces of a column to achieve a pressure balance across the microfluidicdevice. Even if the columns are selected to have the same length anddiameter, temperature and pressures differences between two differentdetectors can disrupt the pressure balancing. When the columns are of adifferent geometry or length, the pressure imbalance can be evengreater.

One possible solution when multiple columns are present is to use aninline restrictor with the column having the highest flow rate, as shownschematically in FIG. 9B. The system of FIG. 9B includes a second column955 fluidically coupled to the first column 910 through the microfluidicdevice 920. Between a first detector 965 and the second column 955 is arestrictor 960. The system shown in FIG. 9B also includes a third column970 fluidically coupled to a detector 975. In this configuration, thecolumn 955 has a higher flow rate than the columns 910 and 970. In theconfiguration shown in FIG. 9B, there are three restrictive zones toconsider: the second column 955 at the oven temperature, the restrictor960 at the oven temperature and the restrictor 960 at the temperature ofthe first detector 965. Equation (9) can be modified to include thesethree zones as shown in Equation (13),

$\begin{matrix}{F_{a} = {\frac{\pi \times T_{a}}{256 \times p_{a}} \times \frac{\left( {p_{i}^{2} - p_{or}^{2}} \right)}{\left( \frac{{T_{r\; 1} \times \eta_{r\; 1} \times L_{r\; 1}} + {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}}{d_{r}^{4}} \right) + \left( \frac{T_{c\; 3} \times \eta_{c\; 3} \times L_{c\; 3}}{d_{c\; 3}^{4}} \right)}}} & (13)\end{matrix}$where L_(c3) is the length of the column 955, η_(c3) is the viscosity ofthe carrier gas in the column 955 and T_(c3) is the absolute temperatureof the column 955. To calculate the length of the restrictor to delivera desired flow rate, Equation (13) can be rearranged to provide Equation(14)

$\begin{matrix}{L_{r} = {\frac{\left\lbrack {{d_{r}^{4} \times \begin{bmatrix}{{\frac{\pi \times T_{a}}{256 \times F_{a} \times p_{a}} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)} -} \\\frac{T_{c\; 3} \times \eta_{c\; 3} \times L_{c\; 3}}{d_{c\; 3}^{4}}\end{bmatrix}} - {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}} \right\rbrack}{T_{r\; 1} \times \eta_{r\; 1}} + L_{r\; 2}}} & (14)\end{matrix}$In use, Equation (11) can be used to calculate the flow rate through thecolumn without the restrictor. Equation (14) can then be used tocalculate the restrictor length to match that flow rate. Also, while notshown, the column 955 can be placed between the restrictor 960 and thefirst detector 965 and Equations (13) and (14) can be modified based onthis rearrangement.

In certain examples, the restrictor internal diameter (or the internaldiameter of other tubing or columns) can be selected to provide adesired flow rate. For a number of GC techniques that use a microfluidicdevice as described herein, it is important to accurately know theinternal diameter of columns and tubes. In practice, the manufacturer'sdescription is assumed to be accurate and is adopted. This can lead tosignificant errors as most relationships involve a calculation based onthe 4th-power of the internal diameter. In these instances, knowledge ofthe true internal diameter would be desirable. Equation (15) can be usedto approximate the flow rate

$\begin{matrix}{F_{a} = \frac{\pi \cdot d_{c}^{4} \cdot T_{a} \cdot \left( {p_{i}^{2} - p_{o}^{2}}\; \right)}{256 \cdot L \cdot p_{a} \cdot \eta \cdot T_{c}}} & (15)\end{matrix}$where F_(a) is the flow rate at the column outlet at ambient temperatureand pressure, d_(c) is the internal diameter of the column, L is thelength of the column, p_(i) is the carrier gas pressure at the columninlet, p_(o) is the outlet pressure, p_(a) is the ambient pressure,T_(c) is the column temperature, T_(a) is the ambient temperature, and ηis the viscosity of the carrier gas at the column temperature. For acolumn or tube at ambient temperature, Equation (15) may be re-arrangedto provide Equation (16).

$\begin{matrix}{F_{a} = {{\left\lbrack \frac{\pi \cdot d_{c}^{4}}{256 \cdot L \cdot p_{a} \cdot \eta} \right\rbrack \cdot \left\lbrack p_{i}^{2} \right\rbrack} - \left\lbrack \frac{\pi \cdot d_{c}^{4} \cdot p_{o}^{2}}{256 \cdot L \cdot p_{a} \cdot \eta} \right\rbrack}} & (16)\end{matrix}$

For a given column or tube, the terms inside the large brackets areconstant and so Equation (16) may be represented as Equation (17).F _(a) =b·└p _(i) ² ┘−a  (17)where a and b are constants. Thus by applying a range of pressures toone end of the column or tube and measuring the flow rate at the other,the value of the constant b can be determined by a least squaresstatistical fit. Once the value of b is established, the internaldiameter may be calculated from Equation (18).

$\begin{matrix}{d_{c} = \sqrt[4]{\frac{256 \cdot L \cdot p_{a} \cdot \eta}{b \cdot \pi}}} & (18)\end{matrix}$As shown specifically in Example 1 below, the diameter of the tubing,e.g., columns, restrictors and the like can accurately be determinedusing these equations. The methods can be implemented in software toprovide a calibration protocol where various diameters of tubing, e.g.,columns, internal tubing, restrictors, etc. can be determined to providefor increased accuracy of the system. The calibration can be performedby the chromatography system or the user can determine the diameter ofthe tubing and enter the calculated diameters into the system for use incontrolling or modulating the flow rates as described herein.

In certain embodiments, to provide for a more user friendly system, theequations noted above may be implemented in software such that a usercan enter the column parameters, e.g., length and internal diameter, theoven temperature and the detector temperature and the system canaccurately predict the particular pressures needed to accomplish adesired separation run. The software can calculate the flow rate,restrictor lengths and/or diameters based on the inputted parameters,and the user can then insert a restrictor having the calculated lengthand diameter at a desired site in the system.

Referring to FIG. 10, an illustrative system including a splittingdevice, which can be a microfluidic device as described herein,configured to split column effluent to two or more detectors is shown.The system 1000 includes an injector 1010 fluidically coupled to apressure regulator 1005 through a supply line 1007. The injector 1010may have a split flow such that a portion of the sample introduced intothe injector 1010 is passed to a column 1020 fluidically coupled to theinjector 1010 through a supply line 1017 and the rest of the sample ispassed along a direction 1015, which may be sent to waste or to anothercolumn, for example. The column 1020 is fluidically coupled to asplitting device 1030 through a supply line 1022, which fluidicallycouples the column 1020 to the splitting device 1030 through an inputport on the splitting device 1030. The splitting device 1030 is alsofluidically coupled to a midpoint pressure regulator 1025 through asupply line 1027. As shown schematically in FIG. 10, the splittingdevice 1030 is configured to split effluent flow from the column 1020into three different flow paths. The splitting device 1030 isfluidically coupled to each of a detector 1050, 1055 and 1060 through aresistor 1035, 1040 and 1045, respectively. During operation of thesystem 1000, column effluent will enter the input port of the splittingdevice 1030 and mix with carrier gas supplied from a midpoint pressureregulator 1025. The effluent can then exit through a plurality of outputports of the splitting device to the detectors 1050, 1055 and 1060.While three detectors are shown in FIG. 10, either fewer, e.g., twodetectors, or more, e.g., four or more detectors, could be used. It isnot necessary to balance the restrictors 1035, 1040 and 1045. Therestrictors may take several forms such as any of those describedherein. By selecting restrictors of appropriate length and internaldiameter, the column effluent may be split between the attacheddetectors over a large range of ratios. The use of a midpoint pressureregulator 1025 in the system 1000, provides some advantageous features.By having a midpoint gas supply, the flow rate into each detector can beincreased according to the needs of each detector thus providing forflexibility in detector flow rates. The low carrier gas flow ratesthrough narrow bore columns can cause even lower flow rates to flow outof the microfluidic device and limit the range of split ratios thatcould be used. The mid-point regulator can provide additional gas flowto overcome these issues and allow very narrow-bore columns to be usedif desired. Column backflushing may also be performed. The midpointregulator also provides for the ability to protect an active MS detectorwhile changing columns.

In certain examples, the system described herein that includes amicrofluidic device can be used in many different configurations. Forexample, it is possible to simultaneously use selective detectors on thesame chromatogram. This feature saves time (only one run needed) andeliminates variations (particularly retention times) between differentchromatograms. One example is the TO-14 US-EPA air monitoring methodwhere both an FID and ECD are used to monitor different compounds in thesame chromatogram. In other configurations, improved dynamic range canbe achieved by splitting different amounts to the same type of detector.Some detectors (e.g. FPD) have a very limited dynamic range and so theability to see large peaks on one detector and small on the other couldbe useful. In some examples as described herein, single columnbackflushing can be performed by controlling the flow rate at variouspoints in the system. This process can save time and eliminate extendedtemperature programs by efficiently removing heavy sample residue fromthe column after the analytes have eluted. Dual column backflushing canalso be performed. For example, one (or more) of the restrictors shownin the system 1000 could be replaced by a GC column. This configurationwould enable the first column to be backflushed while chromatographycontinues on the second column. A mid-point detector can be configuredto monitor the passage of peaks between the two columns to aid setup.Dual column backflushing has a big advantage over single columnbackflushing in that the backflushing occurs simultaneously with thechromatography run thus achieving a substantial time savings. In thecase of air-sensitive detectors, such as an MS or ECD, this system canpermit those detectors to remain at a detection temperature, e.g., hotand active, while a column is being exchanged or an injector isserviced. This feature would save significant time and reduce stress onthe system and so the down time would be minimized. In addition, threeor more column backflushing could also be performed so thatchromatography can proceed on one or more other columns while the first(or more than one column) is being backflushed.

In certain examples, the systems described herein can be used forpolarity tuning. In this technique, the respective residence time ofcompounds within the two columns can be modified by changing themidpoint pressure. This configuration serves to change the effectivepolarity of the combined columns and enables tweaking or fine control ofthe columns selectively to achieve difficult separations.

In accordance with certain examples, one configuration of a microfluidicdevice is shown in FIG. 11. In this cross-sectional view, themicrofluidic device is configured as a wafer 1100 and includes aninternal microchannel 1110 that has a variable diameter at differentportions of the microchannel 1110. For example, the diameter of themicrochannel at area 1125 can be about 300 to about 700 microns, e.g.,about 400 to about 600 microns in diameter, the diameter of themicrochannel at area 1130 can be about 75 microns to about 300 microns,e.g., about 100 microns to about 200 microns in diameter, the diameterof the microchannel at area 1135 can be about 300 to about 700 microns,e.g., about 400 to about 600 microns in diameter, the diameter of themicrochannel at area 1140 can be about 75 microns to about 300 microns,e.g., about 100 microns to about 200 microns in diameter, and thediameter of the microchannel at area 1145 can be about 300 to about 700microns, e.g., about 400 to about 600 microns in diameter. In certainexamples, the diameter of the restricted portion in the microchannel,e.g., area 1140, can be at least two times smaller than the diameter ofthe adjacent channel portions, e.g., at least three times smaller, atleast four times smaller or at least five times smaller, to provide forrestricted fluid flow. The wafer 1100 also includes openings orapertures 1115 and 1120 that can couple the wafer to a wafer holder orother device to hold the wafer 1100 in place during operation of thesystem.

In certain examples, the microfluidic device can include various ports,e.g., inlet and outlet ports, that can provide a fluidic couplingbetween the column and the various other components downstream of themicrofluidic device. One configuration of such a wafer 1200 is shown inFIG. 12. In this configuration, the ports are arranged in series in amicrochannel 1205. A port 1210 is fluidically coupled to a column. Theflow of gas through the wafer 1200 is in the general direction from theport 1210 to ports 1220, 1230, 1240 and 1250. A midpoint pressureregulator can be fluidically coupled to the wafer 1200 at a port 1260.During operation, effluent from the column that enters through the port1210 will be mixed with a carrier gas from the midpoint regulatorentering through the port 1260 and then flow in succession through ports1220, 1230, 1240 and finally 1250. The wafer 1200 can be coupled to aholder through apertures 1265 and 1270. Depending on the exactconfiguration, the various restrictors present in the system may takedifferent operational states. One example of restrictor setup using thewafer 1200 is shown in Table 1.

TABLE 1 Number of Port Detectors 1220 1230 1240 1250 1 Closed ClosedClosed Restrictor 2 Closed Closed Slowest flow Fastest flow restrictorrestrictor 3 Closed Slowest flow Medium flow Fastest flow restrictorrestrictor restrictor 4 Slowest Slowest Fastest Medium Fastest flow flowMedium flow flow restrictor restrictor restrictor restrictorThe restrictors are arranged in order of increasing flow rate with thefastest flow rate being at port 1250. The microchannel can be arrangedso that the outlet ports are within a single microchannel flow path. Toplug or close any particular port, the port may be capped or otherwiseblocked using blanking nuts, fittings, ferrules or other suitabledevices that can provide a fluid tight seal. When closed, desirably noor little dead volume in the port is created that could prevent peaklosses or cause tailing. By using a single wafer as shown in FIG. 12,anywhere from 1-4 detectors can be used without having to change thewafer for each different detector combination.

In accordance with certain examples, the particular length of the flowpath between various ports can vary depending on the desired effect. Oneconfiguration of a wafer having a different flow path configuration isshown in FIG. 13. The wafer 1300 includes a microchannel 1305, a port1310 that is fluidically coupled to a column (not shown), a port 1360that is fluidically coupled to a midpoint pressure regulator (not shown)and ports 1320, 1330, 1340 and 1350, each of which may or may not befluidically coupled to a restrictor and/or detector. A portion of themicrochannel 1305 is elongated to provide an increased flow path lengthto permit additional mixing of carrier gas from the midpoint regulatorport 1360 and the column effluent port 1310. Such increased length can,for example, provide additional time to provide increased sampleresidence time and a more homogenous mixture of column effluent andcarrier gas, which can be used to avoid or reduce diffusional broadeningof the analyte peaks as described in more detail herein. In addition,the particular length of the flow path between any two or more of ports1320, 1330, 1340 and 1350 can be different than the other lengths. Whensuch a different length is present, it may be desirable to alter therestrictor flow rate to balance the flow rates of gas provided to thedifferent detectors. Openings 1365 and 1370 can be used, for example, toattach the microfluidic device to a holder or other device designed toretain the microfluidic device at a desired site or in a desiredorientation.

It will be recognized by the person of ordinary skill in the art, giventhe benefit of this disclosure that the exact number of ports in thewafer can vary and may be, for example, fewer ports or more ports thatthe illustrative configurations shown in FIGS. 12 and 13. Illustrativeconfigurations are shown in FIGS. 14A-15B. Referring to FIG. 14A, awafer includes a column effluent port 1405 fluidically coupled to amidpoint pressure regulator port 1425 and ports 1410 and 1415 each ofwhich can be fluidically coupled to a restrictor and/or detector, forexample. Apertures 1430 and 1435 can be used to attach the wafer to aholder or other structures of the microfluidic device. The length of theflow path between the two ports 1410 and 1415 can vary, and theparticular restrictor flow rate can be altered to provide substantiallythe same flow rate through the different ports, if desired. FIG. 14Bshows a configuration where the length of the flow path between ports1410 and 1415 has been lengthened. Such lengthening may be desirable,for example, to provide more spacing for coupling of fittings to thevarious ports and facilitate overall setup of the device, to provide forincreased residence time or other desired performance.

While two ports 1410 and 1415 are shown in FIGS. 14A and 14B, port 1415can be omitted and a single port may be present. In the alternative, oneor more additional ports can be present to provide fluidic couplingbetween such additional port(s) and a restrictor(s) and/or detector(s).Two configurations using additional ports are shown in FIGS. 15A and15B. Referring to FIG. 15A, the wafer includes a column effluent port1505 fluidically coupled to a midpoint pressure regulator port 1510 andto ports 1520, 1525, 1530, 1535, 1540 and 1545. Apertures 1550 and 1555can be used to attach the wafer to a holder or other portions of themicrofluidic device. FIG. 15B shows a similar arrangement to that ofFIG. 15A, but the position of port 1535 has been moved.

In certain examples, the exact cross-sectional shape and angles of themicrochannels can vary. In certain examples, the cross-sectional shapeof the microchannel is circular or substantially circular, whereas inother examples, elliptical shapes or other non-circular shapes can bepresent. Similarly, the angle of the microchannel between two or moreports can vary and where a non-continuous flow path is present, theangle made by the change in direction of the flow path may be a sharpangle or may be a gradual angle such as, for example, an elbow or acurved surface. For example, in fluid chromatography systems where sharpangles may create turbulent flow, the angles can be configured as elbowor gradual turns to avoid or reduce such turbulence.

In certain examples, the microfluidic devices described herein can beused in many different configurations. FIGS. 16-25 show severalillustrative configurations. Referring to FIG. 16, a single detectorconfiguration that can be used, for example, to backflush and in a MSVent mode is shown. The system 1600 includes an injector 1610fluidically coupled to a pressure regulator 1605 through a supply line1607. The injector 1610 is also fluidically coupled to a column 1620through a supply line 1612. The column 1620 is fluidically coupled to amicrofluidic device 1625 through a supply line 1617. The microfluidicdevice 1625 includes a column effluent port 1627 fluidically coupled toa midpoint pressure regulator 1630 through a port 1633. Gas is providedfrom the midpoint pressure regulator 1630 to the port 1633 through asupply line 1632. The microfluidic device 1625 includes ports 1635,1640, 1645 and 1650. In the embodiment of FIG. 16, ports 1635, 1640 and1645 are closed or plugged such that no gas flows into them. The port1650 is fluidically coupled to a detector 1660 through a restrictor1655. In operation, a sample is introduced into the injector 1610 andspecies in the sample can be separated using the column 1620. Specieselute from the column 1620 and are provided to the detector 1660 throughthe microfluidic device 1625. Flow control of the overall system may beperformed as described herein or using other suitable algorithms. Thearrows show the general gas flow in the system 1600. If desired, any ofports 1635, 1640 or 1645 can be coupled to a sniffer or other device toprovide for in-line sampling of gas and/or species in the gas within themicrochannel of the microfluidic device 1625. In addition, by increasingthe flow of gas provided by the midpoint pressure regulator to begreater than the flow rate to the column, the system 1600 can bebackflushed or can be vented, e.g., can be operated in a MS vent mode.

In accordance with certain examples and referring to FIG. 17, a dualdetector configuration is shown. The system 1700 includes an injector1710 fluidically coupled to a pressure regulator 1705 through a supplyline 1707. The injector 1710 is also fluidically coupled to a column1720 through a supply line 1712. The column 1720 is fluidically coupledto a microfluidic device 1725 through a supply line 1717. Themicrofluidic device 1725 includes a column effluent port 1727fluidically coupled to a midpoint pressure regulator 1730 through a port1733. Gas is provided from the midpoint pressure regulator 1730 to theport 1733 through a supply line 1732. The microfluidic device 1725includes ports 1735, 1740, 1745 and 1750. In the embodiment of FIG. 17,ports 1735 and 1740 are closed or plugged such that no gas flows intothem. The ports 1745 and 1750 are each fluidically coupled to a detector1770 and 1760, respectively, through a restrictor 1765 and 1755,respectively. In operation, a sample is introduced into the injector1710 and species in the sample can be separated using the column 1720.Species elute from the column 1720 and are provided to one or both ofthe detectors 1760 and 1770 through the microfluidic device 1725. Flowcontrol of the overall system may be performed as described herein orusing other suitable algorithms. The arrows show the general gas flow inthe system 1700. The detectors 1760 and 1770 may be the same or may bedifferent. In addition different peaks can be provided to differentdetectors by including suitable valving in the supply lines and/or byactuating one of more of the ports of the microfluidic device 1725 to bein a closed position, e.g., using a switching valve.

In accordance with certain examples and referring to FIG. 18, a threedetector configuration is shown. The system 1800 includes an injector1810 fluidically coupled to a pressure regulator 1805 through a supplyline 1807. The injector 1810 is also fluidically coupled to a column1820 through a supply line 1812. The column 1820 is fluidically coupledto a microfluidic device 1825 through a supply line 1822. Themicrofluidic device 1825 includes a column effluent port 1827fluidically coupled to a midpoint pressure regulator 1830 through a port1833. Gas is provided from the midpoint pressure regulator 1830 to theport 1833 through a supply line 1832. The microfluidic device 1825includes ports 1835, 1840, 1845 and 1850. In the embodiment of FIG. 18,port 1835 is closed or plugged such that no gas flows into it. The ports1840, 1845 and 1850 are each fluidically coupled to a detector 1880,1870 and 1860, respectively, through a restrictor 1875, 1865 and 1855,respectively. In operation, a sample is introduced into the injector1810 and species in the sample can be separated using the column 1820.Species elute from the column 1820 and are provided to one or more ofthe detectors 1860, 1870 and 1880 through the microfluidic device 1825.Flow control of the overall system may be performed as described hereinor using other suitable algorithms. The arrows show the general gas flowin the system 1800. The detectors 1860, 1870 and 1880 may be the same ormay be different or two of the detector 1860, 1870 and 1880 may be thesame. In addition different peaks can be provided to different detectorsby including suitable valving in the supply lines and/or by actuatingone of more of the ports of the microfluidic device 1825 to be in aclosed position, e.g., using a switching valve.

In accordance with certain examples and referring to FIG. 19, a system1900 including four detectors is shown. The system 1900 includes aninjector 1910 fluidically coupled to a pressure regulator 1905 through asupply line 1907. The injector 1910 is also fluidically coupled to acolumn 1920 through a supply line 1912. The column 1920 is fluidicallycoupled to a microfluidic device 1925 through a supply line 1922. Themicrofluidic device 1925 includes a column effluent port 1927fluidically coupled to a midpoint pressure regulator 1930 through a port1933. Gas is provided from the midpoint pressure regulator 1930 to theport 1933 through a supply line 1932. The microfluidic device 1925includes ports 1935, 1940, 1945 and 1950. In the embodiment of FIG. 19,the ports 1935, 1940, 1945 and 1950 are each fluidically coupled to adetector 1990, 1980 and 1970 and 1960, respectively, through arestrictor 1985, 1975, 1965 and 1955, respectively. In operation, asample is introduced into the injector 1910 and species in the samplecan be separated using the column 1920. Species elute from the column1920 and are provided to one or more of the detectors 1960, 1970, 1980and 1990 through the microfluidic device 1925. Flow control of theoverall system may be performed as described herein or using othersuitable algorithms. The arrows show the general gas flow in the system1900. The detectors 1960, 1970, 1980 and 1990 may be the same or may bedifferent or any two or three of the detectors 1960, 1970, 1980 and 1990may be the same. In addition different peaks can be provided todifferent detectors by including suitable valving in the supply linesand/or by actuating one of more of the ports of the microfluidic device1925 to be in a closed position, e.g., using a switching valve.

In accordance with certain examples and referring to FIG. 20, a system2000 including a single detector 2070 and a sniffer port 2060 is shown.The system 2000 includes an injector 2010 fluidically coupled to apressure regulator 2005 through a supply line 2007. The injector 2010 isalso fluidically coupled to a column 2020 through a supply line 2012.The column 2020 is fluidically coupled to a microfluidic device 2025through a supply line 2022. The microfluidic device 2025 includes acolumn effluent port 2027 fluidically coupled to a midpoint pressureregulator 2030 through a port 2033. Gas is provided from the midpointpressure regulator 2030 to the port 2033 through a supply line 2032. Themicrofluidic device 2025 includes ports 2035, 2040, 2045 and 2050. Inthe embodiment of FIG. 20, the ports 2035 and 2040 are closed or pluggedsuch that no gas flows into them. The port 2045 is fluidically coupledto the detector 2070 through a restrictor 2065. The port 2050 isfluidically coupled to the sniffer port 2060, which can be used forin-line sampling or monitoring of species in the effluent or generallyprovides a port from which species in the fluid path can be withdrawn,if desired, through a restrictor 2055. In operation, a sample isintroduced into the injector 2010 and species in the sample can beseparated using the column 2020. Species elute from the column 2020 andare provided to one or more of the detector 2070 or the sniffer port2060 through the microfluidic device 2025. Flow control of the overallsystem may be performed as described herein or using other suitablealgorithms. The arrows show the general gas flow in the system 2000. Thesniffer port 2060 may typically remain in a closed position until theuser desires to sample from that port. Suitable valving in the supplylines and/or by actuating the sniffer port 2060 can open the snifferport, as desired.

In accordance with certain examples and referring to FIG. 21, a system2100 that is configured for backflushing or venting in an MS system isprovided. The system 2100 is similar to that shown in FIG. 17, but theflow rates of the various gases are altered to perform the backflushingor venting. Referring to FIG. 21, system 2100 includes an injector 2110fluidically coupled to a pressure regulator 2105 through a supply line2107. The injector 2110 is also fluidically coupled to a column 2120through a supply line 2112. The column 2120 is fluidically coupled to amicrofluidic device 2125 through a supply line 2122. The microfluidicdevice 2125 includes a column effluent port 2127 fluidically coupled toa midpoint pressure regulator 2130 through a port 2133. Gas is providedfrom the midpoint pressure regulator 2130 to the port 2133 through asupply line 2132. The microfluidic device 2125 includes ports 2135,2140, 2145 and 2150. In the embodiment of FIG. 21, ports 2135 and 2140are closed or plugged such that no gas flows into them. The ports 2145and 2150 are each fluidically coupled to a detector 2170 and a detector2160, respectively, through a restrictor 2165 and 2155, respectively. Inoperation, a sample is introduced into the injector 2110 and species inthe sample can be separated using the column 2120. Species elute fromthe column 2120 and are provided to one or both of the detectors 2160and 2170 through the microfluidic device 2125. Flow control of theoverall system may be performed as described herein or using othersuitable algorithms. The arrows show the general gas flow in the system2100 in this backflushing configuration. The detectors 2160 and 2170 maybe the same or may be different. In the backflushing or venting mode,the flow rate of gas from the midpoint pressure regulator 2130 isgreater than the flow of gas from the pressure regulator 2105, e.g., p₂is greater than p₁. The result of this differential flow is that gas isflushed back through the column 2120 and can be vented from the system,for example, through outlet 2102. Where an MS detector is present, thedifferential fluid flow can be used to maintain the MS detector at itsoperating temperature while the system can be flushed. Such an advantagecan provide a substantial time savings.

In accordance with certain examples and referring to FIG. 22, a dualcolumn backflush configuration is shown. The system 2200 includes aninjector 2210 fluidically coupled to a pressure regulator 2205 through asupply line 2207. The injector 2210 is also fluidically coupled to afirst column 2220 through a supply line 2212. The first column 2220 isfluidically coupled to a microfluidic device 2225 through a supply line2222. The microfluidic device 2225 includes a column effluent port 2227fluidically coupled to a midpoint pressure regulator 2230 through a port2233. Gas is provided from the midpoint pressure regulator 2230 to theport 2233 through a supply line 2232. The microfluidic device 2225includes ports 2235, 2240, 2245 and 2250. In the embodiment of FIG. 22,ports 2235, 2240 and 2245 are closed or plugged such that no gas flowsinto them. The port 2250 is fluidically coupled to a second column 2255.The second column 2255 is fluidically coupled to a detector 2260. Inoperation, a sample is introduced into the injector 2210 and species inthe sample can be separated using the first column 2220. Species elutefrom the first column 2220 and are provided to the second column 2255through the microfluidic device 2225. Flow control of the overall systemmay be performed as described herein or using other suitable algorithms.The arrows show the general gas flow in the system 2200. Theconfiguration of system 2200 permits backflushing of the first column2220 once the effluent enters into the microfluidic device 2225. Byincreasing the pressure p₂ to be larger than the pressure p₁, e.g., sothe flow rate from the midpoint pressure regulator 2230 is greater thanthe flow rate from the pressure regulator 2205, gas will flow into thefirst column 2220 to backflush it, and will also pass the effluent fromthe microfluidic device 2225 to the second column 2255 for continued oradditional separation using the second column 2255. Polarity tuningmethods may also be performed using a system as shown in FIG. 22.

In accordance with certain examples, a dual column backflushconfiguration with a midpoint monitoring detector is shown in FIG. 23.The system 2300 includes an injector 2310 fluidically coupled to apressure regulator 2305 through a supply line 2307. The injector 2310 isalso fluidically coupled to a first column 2320 through a supply line2312. The first column 2320 is fluidically coupled to a microfluidicdevice 2325 through a supply line 2322. The microfluidic device 2325includes a column effluent port 2327 fluidically coupled to a midpointpressure regulator 2330 through a port 2333. Gas is provided from themidpoint pressure regulator 2330 to the port 2333 through a supply line2332. The microfluidic device 2325 includes ports 2335, 2340, 2345 and2350. In the embodiment of FIG. 23, ports 2335 and 2340 are closed orplugged such that no gas flows into them. The port 2350 is fluidicallycoupled to a second column 2355. The second column 2355 is fluidicallycoupled to a detector 2360. The port 2345 is fluidically coupled to adetector 2370 through a restrictor 2365. In operation, a sample isintroduced into the injector 2310 and species in the sample can beseparated using the first column 2320. Species elute from the firstcolumn 2320 and are provided to the second column 2355 through themicrofluidic device 2325. In addition, species can be detected by usingthe detector 2370. Flow control of the overall system may be performedas described herein or using other suitable algorithms. The arrows showthe general gas flow in the system 2300. The configuration of system2300 permits backflushing of the first column 2320 once the effluententers into the microfluidic device 2325. By increasing the pressure p₂to be larger than the pressure p₁, gas will flow into the first column2320 to backflush it, and will also pass the effluent from themicrofluidic device 2325 to the second column 2355 for continued oradditional separation using the second column 2355. Effluent can also beprovided to the detector 2370 without any further separation. Polaritytuning methods may also be performed using a system as shown in FIG. 23.

In accordance with certain examples, a three column backflushconfiguration is shown in FIG. 24. The system 2400 includes an injector2410 fluidically coupled to a pressure regulator 2405 through a supplyline 2407. The injector 2410 is also fluidically coupled to a firstcolumn 2420 through a supply line 2412. The first column 2420 isfluidically coupled to a microfluidic device 2425 through a supply line2422. The microfluidic device 2425 includes a column effluent port 2427fluidically coupled to a midpoint pressure regulator 2430 through a port2433. Gas is provided from the midpoint pressure regulator 2430 to theport 2433 through a supply line 2432. The microfluidic device 2425includes ports 2435, 2440, 2445 and 2450. In the embodiment of FIG. 24,ports 2435 and 2440 are closed or plugged such that no gas flows intothem. The port 2450 is fluidically coupled to a second column 2455. Thesecond column 2455 is fluidically coupled to a detector 2460. The port2445 is fluidically coupled to a third column 2465 that is fluidicallycoupled to a detector 2470. In operation, a sample is introduced intothe injector 2410 and species in the sample can be separated using thefirst column 2420. Species elute from the first column 2420 and areprovided to the second column 2455 and third column 2465 through themicrofluidic device 2425. In addition, species can be detected by usingthe detectors 2460 and 2470. Flow control of the overall system may beperformed as described herein or using other suitable algorithms. Thearrows show the general gas flow in the system 2400. The configurationof system 2400 permits backflushing of the first column 2420 once theeffluent enters into the microfluidic device 2425. By increasing thepressure p₂ to be larger than the pressure p₁, gas will flow into thefirst column 2420 to backflush it, and will also pass the effluent fromthe microfluidic device 2425 to the second column 2455 and third column2465 for continued or additional separation using these additionalcolumns. Polarity tuning methods may also be performed using a system asshown in FIG. 24. While not shown, the system of FIG. 24 may include adetector fluidically coupled to one of the ports 2435 or 2440.

In certain examples and referring to FIG. 25, a system may be configuredwith a microfluidic device to split the injector flow into two or morecolumns. The system 2500 includes an injector 2510 fluidically coupledto a pressure regulator 2505 through a supply line 2507. The injector2510 is also fluidically coupled to a microfluidic device 2525 through asupply line 2512. The microfluidic device 2525 includes a port 2527fluidically coupled to a midpoint pressure regulator 2530 through a port2533. Gas is provided from the midpoint pressure regulator 2530 to theport 2533 through a supply line 2532. The microfluidic device 2525includes ports 2535, 2540, 2545 and 2550. In the embodiment of FIG. 25,ports 2535 and 2540 are closed or plugged such that no gas flows intothem. The port 2550 is fluidically coupled to a first column 2555. Theport 2545 is fluidically coupled to a second column 2565. Each of thecolumns 2555 and 2565 is coupled to a detector 2560 and 2570,respectively. In operation, a sample is introduced into the injector2510 and species in the sample can be separated using the first column2555 and the second column 2565. The column media in columns 2555 and2565 can be the same or can be different. Species elute from the columnsand are passed to their respective detectors for detection. Flow controlof the overall system may be performed as described herein or usingother suitable algorithms. The arrows show the general gas flow in thesystem 2500. While not shown, the system of FIG. 25 may include adetector fluidically coupled to one of the ports 2535 or 2540. Arestrictor or another column may be positioned between port 2535 or port2540 and a detector.

In certain examples, while the systems described above include amicrofluidic device that is designed to be coupled to a midpointpressure regulator, there are applications that are cost sensitive or donot need any gas added to the column effluent. When such applicationsare performed, a different microfluidic device can be used. Or in thealternative, the midpoint pressure port of the wafers shown in FIGS.11-15B can be blocked or capped such that no gas flow can enter. Onesuch configuration where the midpoint pressure port is omitted is shownin FIG. 26A. The microfluidic device 2600 includes serial ports 2610,2615, 2620, 2625, 2630 and 2635. The microfluidic device is scalable inthat all or fewer than all of the ports can be used. Apertures 2640 and2645 may be used to attach the microfluidic device 2600 to a holder orother device. The port 2615 is downstream of where the column effluententers the microfluidic device at the port 2610. The port 2615 may beconnected to a gas inlet or this port can be capped or blocked. Each ofthe ports 2620, 2625, 2630 and 2635 may be fluidically coupled to acolumn, restrictor, detector and various combinations thereof. Wherefewer than four couplings are desired, any one or more of the ports canbe capped or plugged to shut that port off. In addition, a microfluidicdevice having fewer than six ports can be designed. One such example isshown in FIG. 26B. The microfluidic device 2650 includes ports 2655,2660, 2665 and 2670. Apertures 2675 and 2680 may be used to attach thewafer 2650 to a holder or other device. The ports 2660, 2665 and 2670can be fluidically coupled to a column, restrictor, detector and variouscombinations thereof. In one alternative, the port 2660 can befluidically coupled to a gas source to provide additional gas throughthe wafer 2650. Other port numbers, configuration and geometriesconsistent with the microfluidic devices 2600 and 2650 may also be useddepending on the desired number of detectors to be used in the system orthe particular desired configuration of the system.

In accordance with certain examples, the microfluidic devices describedherein include one or more microchannels in the wafer. The exactconfiguration of the microchannel and how such microchannels areproduced can vary depending on the particular material selected for useas a wafer. For example, the microchannel can be chemically etched,laser etched, drilled, grinded or molded into the wafer duringproduction. The widths and overall geometry of the microchannels mayvary. In one embodiment, the width of the microchannels can vary fromabout 10 microns to about 750 microns, for example, 50 microns to about500 microns, for example, about 10 microns to about 100 microns, about100 microns to about 300 microns, or about 300 microns to about 500microns. The cross-sectional geometry of the microchannel may becircular, elliptical, triangular or other geometries. As discussedherein, it is desirable, but not required, that the microchannels havesmooth transitions, e.g., elbows and the like, to facilitate gas flowthrough the microchannels.

In certain examples, the microfluidic device can be used in a multilayerdevice or a multicomponent device. For example, the microfluidic devicecan be sandwiched between two or more other devices to provide for asubstantially fluid tight seal to prevent leaks. One or more gaskets orgasket materials can be used to further enhance the seal. Additionally,gaskets, tapes or other materials can be used at the ports of the deviceto provide additional sealing, if desired. In some examples, themicrofluidic device can be a multi-layer structure itself, e.g., alaminated wafer, with sequential additions of layers being added to formthe microchannels. One example of a microfluidic device and two platesused to hold the wafer is shown in FIG. 27. The wafer 2710 can besandwiched between a first plate 2720 and a second plate 2730. A ferruleor fitting 2740 can be attached to the assembly to hold the microfluidicdevice 2710 and plates 2720, 2730 together during use of themicrofluidic device.

In certain examples where the microfluidic device is configured as awafer, the wafer can be produced from various materials includingmetals, plastics, composites, polymers, steels, stainless steels,alloys, and other materials that can be assembled to providemicrochannels. For example, various layers of the wafer can be producedusing stainless steel plates that can be laminated or welded together toform an overall microchannel structure within the wafer. In certainembodiments, layers of polyethertherketone or other polymers having adesired channel portion etched, drilled or otherwise carved into it canbe laser or solvent welded to each other to provide the wafer.Regardless of the particular material selected for use in the wafer, thematerial desirably is inert such that no unwanted chemical reactionswill occur between the sample and the wafer. In examples where the wafermaterial may be reactive, the microchannels (or the entire wafersurface) can be coated with an inert material such as, for example,polytetrafluoroethylene or other generally inert materials. Where thesample to be analyzed is corrosive, the microchannels (or the entirewafer surface) can be coated with yttria, alumina, or other materialsthat are resistant to corrosion and can protect the underlying waferstructure from damage. If a coating is used, the coating should be thickenough and robust enough to avoid leaching off, flaking or desorbing,which could lead to interference with the sample measurements. Inaddition, the materials used in the microfluidic device are desirablyheat tolerant such that they do not melt or experience any substantialthermal deformation when used in a hot oven, such as those ovens andtemperatures commonly encountered and used in chromatography systemseparations.

In accordance with certain examples, the restrictors that can be usedwith the devices and systems disclosed herein may vary in configurationand design. In certain examples, the microfluidic devices describedherein can include a by-pass restrictor or other comparable device toreduce or restrict flow of gas and/or sample into unused areas of themicrofluidic device. One such example is shown in FIG. 28. Themicrofluidic device 2800 includes a plurality of ports 2810, 2815, 2820,2825, 2830, and 2835. The port 2810 provides effluent from the column.The port 2815 can be fluidically coupled to a first detector (optionallywith an in-line restrictor). The port 2820 is fluidically coupled to aswitching gas source. The port 2825 is not used in this configuration.The port 2830 is fluidically coupled to another switching gas source.The port 2835 is fluidically coupled to a second detector (optionallywith an in-line restrictor). A fluid connection 2850, e.g., internal orexternal, may be provided between the ports 2820 and 2830 to by-pass theport 2825. The fluid connection 2850 may include an external needlevalve or may include a valve such as, for example, a switching valvesuch as a solenoid valve. The bypass dimensions can be adjusted toensure that substantially no diffusion of gas occurs into an unusedportion of the wafer. In operation, sufficient gas flow is provided tothe by-pass restrictor to prevent sample diffusion along the switchinggas inlet channel not in use at a particular point in time. The flowrate is desirably low to avoid or reduce the volume of gas entering intothe GC column, which can dilute the sample.

In certain examples, to reduce the flow rate through the microchannels,the switching gas channels can be narrowed, tapered or constricted nearthe ends to increase the gas velocity as it enters into the sample flowpath. For example and referring to FIG. 28, at or near the union ofmicrochannel sections 2852 and 2854, the diameter of the microchannelsection 2854 may be less than that of the microchannel section 2852. Forexample, if the microchannel section 2852 is about 600 microns indiameter, the microchannel section 2854 can be reduced to 300 microns indiameter. It will be recognized by the person of ordinary skill in theart, given the benefit of this disclosure, that a 50% reduction is notrequired. Other percentages and ratios may be used. For example, theratio of the unconstricted microchannel section diameter:constrictedmicrochannel section diameter can be about 5:1 to about 1.1:1, e.g.,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, moreparticularly about 3:1 to about 1.1:1, for example about 2.5:1 to about1.2:1 or about 2:1 to about 1.5:1. In some examples, the overalldiameter of the microchannel can be about 400-500 microns andconstricted portions of the microchannel can have diameters of about100-200 microns. The exact length of the bypass restrictor channel canalso vary with illustrative lengths of about 5 mm to about 30 mm, moreparticularly about 10 mm to about 20 mm, e.g., about 11, 12, 13, 14, 15,16, 17, 18, 19 mm or any value in between these specific lengths.

In accordance with certain examples, in assembly and use of themicrofluidic devices described herein, the microfluidic device istypically sandwiched or encased in a multicomponent device to provide amicrofluidic device that can be coupled to pneumatic tubing in a GCsystem. As described herein, these systems can be used in many differentconfigurations and in multi-dimensional chromatographic analyses. Inaddition, while certain embodiments of the microfluidic devices aredescribed herein, it will be recognized by the person of ordinary skillin the art, given the benefit of this disclosure, that the microfluidicdevices can be used in combination with each other, e.g., by mountingthem back to back in the same system. Suitable fluid connections todesired ports may be provided using pneumatic tubing and otherconnectors. In addition, crossover channels, e.g., either within onemicrofluidic device or between two or more different microfluidicdevices can be provided. In-line valves or actuators can be used tocontrol the gas flow to a desired port and/or to a desired microfluidicdevice. For example, a solenoid valve can be modulated, e.g., at about10-100 Hz, e.g., about 50 Hz, to permit flow of one species to a desiredport or detector. The solenoid valve may be closed or switched to stopsuch flow to a particular port. Some of these configurations aredescribed in detail below.

In certain examples and referring to FIG. 29, one example of a systemincluding a microfluidic device with a crossover switch is shown. Use ofa crossover switch may be particularly desirable where, for example,column switching, automated screening, backflushing, large volumeinjections, multi-dimensional chromatography or multiplexing operationsare desired. For example, one MS detector can simultaneously receivesample from two different columns. The system 2900 includes an injector(not shown) fluidically coupled to a first column 2905. The first column2905 is fluidically coupled to a microfluidic device 2910 through afluid flow path, e.g., pneumatic tubing. The system also includes aplurality of restrictors 2915, 2920, 2925 and 2930 each fluidicallycoupled to the microfluidic device 2910. As discussed herein, therestrictors can be used to balance the pressure in the system. Themicrofluidic device 2910 includes a switching valve that is operative toprovide a crossover path such that species from the first column 2905and a second column 2940, which can be fluidically coupled to its owninjector (not shown), can selectively be provided to a detector 2935 ora detector 2945. For example, by modulating the switching valve, a fluidflow path can be provided between two or more desired components of thesystem and can provide different species eluting from either column toone or both of the detectors. In addition, the sample flow can be splitsuch that effluent from one column is provided to both of the detectors2935 and 2945, as described herein. The various pressures in the systemcan be balanced as described herein or using other suitableconfigurations. It is desirable that the columns have the same internaldiameter but the lengths can be different. The injectors can be liquidinjectors or other suitable injectors, e.g., high speed injectors,automatic thermal desorption injectors and other injectors commonly usedwith GC devices and systems. The detectors 2935 and 2945 may be the sameor may be different. Desirably, one of the detectors can be a MSdetector and the other may be a different detector such as, for example,those described herein.

In certain embodiments, two or more microfluidic devices can be used inthe illustrative embodiment of FIG. 29. For example, a crossoverconnection can be provided between two or more microfluidic devices eachfluidically coupled to at least one column. For example, a firstmicrofluidic device can be fluidically coupled to the first column 2905,and a second microfluidic device can be fluidically coupled to thesecond column 2940. The switching valve can be actuated to providedesired flow to a particular port of the microfluidic device or toprovide flow between the microfluidic devices.

Various possible connections between the components of FIG. 29 are shownin more detail in FIGS. 30A and 30B. Referring to FIG. 30A, theswitching valve of the microfluidic device 3015 may be configured suchthat effluent from a column 3010 is provided to a detector 3025. Aninjector 3005 is fluidically coupled to the column 3010. An in-linerestrictor 3020 is between the microfluidic device 3015 and the detector3025. An injector 3030 provides sample to a second column 3035. Themicrofluidic device 3015 is also fluidically coupled to the secondcolumn 3035. The microfluidic device 3015 is fluidically coupled to asecond detector 3045 through a restrictor 3040. In the configurationshown in FIG. 30A, the microfluidic device 3015 is configured such thateffluent from the column 3010 is provided to the detector 3025 andeffluent from the column 3035 is provided to the detector 3045. In thecrossover configuration shown in FIG. 30B, effluent from the column 3035is provided to the detector 3025 and effluent from the column 3010 isprovided to the detector 3045. In operation of the system, themicrofluidic device may be operated between these two different statessuch that certain species from the column 3010 can be provided to thedetector 3025 and other species from the column 3010 can be provided tothe detector 3045. Similar operations may be performed for speciesexiting the column 3035. If desired, sample from the different columns3010, 3035 can be provided to the same detector using the microfluidicdevice. In addition, more than one microfluidic device can be used inthe system of FIG. 30, if desired.

In certain examples, the microfluidic device 3015 can also be used tobackflush one or both of the columns 3010 and 3035. For example, bylowering the gas flow from pressure regulators p₁ and p₂ to be less thanthe flow from pressure regulator p₃, both of the column 3010 and 3035can be backflushed for cleaning. In an alternative configuration, onlythe pressure p₁ or p₂ can be lowered to be less than p₃ such that onlyone of the columns is backflushed and separation may continue using theother column.

In certain examples, one of the detectors shown in FIGS. 30A and 30B maybe omitted or substituted with another device such as another column, avent or other components commonly used in chromatography systems. Oneconfiguration is shown in FIG. 31. The system 3100 includes an injector3105 fluidically coupled to a first column 3110. The column 3110 isfluidically coupled to a microfluidic device 3115. A vent 3125 is influid communication with the microfluidic device 3115 through arestrictor 3120. A second injector 3130 is fluidically coupled to asecond column 3135. The second column 3135 is fluidically coupled to themicrofluidic device 3115. The microfluidic device 3115 is alsofluidically coupled to a detector 3145 through a restrictor 3140. Inoperation of the system 3100, the microfluidic device can be used toprovide column effluent from both the column 3110 and the column 3135 tothe detector 3145. Where species eluting from the column are not desiredfor analysis, the microfluidic device can be used to divert thosespecies to the vent 3125. In an alternative configuration, the vent 3125permits venting of the system while permitting the detector 3145 to bemaintained at normal operating temperature and pressure. This feature isparticularly desirable where the detector 3145 is a mass spectrometer.

In accordance with certain examples, one illustration of a microfluidicdevice configured as a wafer and including a crossover flow path isshown in FIGS. 32A-32D. The overall wafer construct is shown in FIG. 32Awith various layers shown exploded in FIGS. 32B-32D. Referring to FIG.32A, the wafer 3200 generally includes a multilayer substrate 3205having one or more microchannels therein. In this embodiment, themicrochannel has six ports 3210, 3215, 3220, 3225, 3230, and 3235. Theport 3210 can be an inlet port from a first column, the port 3230 can bea port for a first switching gas, the port 3220 can be a port for asecond switching gas, the port 3235 can be an outlet port to a firstdetector, the port 3225 can be an inlet port from a second column, andthe port 3215 can be an outlet port to a second detector (or vent). Thedevice includes crossover channels 3242 and 3244 and constrictedchannels 3246 and 3247. In producing the wafer, different layers can beassembled to provide the various flow paths. Referring to FIG. 32B, onelayer can provide the crossover path 3242, which provides fluidiccoupling between ports 3215 and 3230. The layer shown in FIG. 32B can bethe bottom layer of the laminate or a layer on an external surface ofthe laminate depending on the exact orientation of the microfluidicdevice. Another layer (see FIG. 32D), can provide the crossover path3244 and the constricted channels 3246 and 3247. The layer shown in FIG.32D can be the top layer of the laminate or a layer on an externalsurface of the laminate opposite to the layer shown in FIG. 32B with themiddle layer positioned between the top layer and the bottom layer. Thecrossover path 3244 can provide for fluidic coupling between the ports3220 and 3235. The constricted channel 3246 provides fluidic couplingbetween the ports 3230 and 3235. The constricted channel 3247 providesfluidic coupling between the ports 3215 and 3220. The middle layer (FIG.32C) may include suitable flow paths to complete the microchannel and toprovide fluid flow between desired ports of the microfluidic device.When the various layers are laminated together to provide a wafer, thefluid flow paths will be produced and can be used to control thedirection of species in the chromatographic system. The overall wafercan be mounted to a sample holder or other suitable device usingapertures 3250 and 3255 and suitable fittings, e.g., screws, nuts,bolts, ferrules, etc. Each of the various layers shown in FIGS. 32B-32Dmay itself be a multilayer structure or laminate or may be a generallysolid body having respective microchannels etched or otherwise includedtherein. Gaskets, sealants or other materials may be added between thelayers to facilitate a fluid tight seal to avoid or reduce thelikelihood that internal leaks may occur.

In certain examples and as discussed herein, the microfluidic device mayinclude one or more actuators or switching valves that can couple ordecouple two or more fluid flow paths. The position of the actuatorprovides for fluid flow between two or more ports or prevents fluid flowbetween two or more ports. The microfluidic device may include a lowcost solenoid valve that can be opened, closed or modulated at a desiredfrequency to connect two or more flow paths or to stop flow between twoor more flow paths. In some examples, the solenoid valve can be actuatedbetween a fully open and a fully closed position. The frequency withwhich the solenoid is actuated depends on the particular type ofchromatography being performed, e.g., heartcut or solvent dump, theparticular ports to be connected and the desired effect on pressure thatcan be accomplished by opening and closing the valve, and illustrativefrequencies include, but are not limited to 5-200 Hz, 10-100 Hz, 20-90Hz, 30-80 Hz, 40-70 Hz, 45-65 Hz, and 50-60 Hz. The solenoid valves aretypically external to the wafer and coupled to a desired port throughpneumatic tubing or other suitable connections. In some examples, theswitching valve may be integrated into the port of the wafer to providefor fewer components for the end user to connect.

In certain embodiments, two or more serially connected switching valvescan be used which are the same or are different. For example, a solenoidvalve in-line with a proportional valve can be fluidically coupled to aport of the microfluidic device. The system may include gas flowmonitors, pressure transducers or other devices to ensure that thepressure in the system is balanced.

In certain examples, the switching valve can be controlled using acontroller, processor or other suitable electrical components. Oneconfiguration that can be used to modulate the valve is shown in FIGS.33A and 33B. Referring to FIG. 33A, a function generator 3310 iselectrically coupled to a transistor driver 3320 and is operative toprovide a desired waveform to the transistor driver 3320. The transistordriver 3320 is electrically coupled to a solenoid valve 3330 and isoperative to modulate the solenoid valve 3330 at a frequencycorresponding to the particular waveform provided by the functiongenerator 3310. The waveform provided by the function generator 3310 canvary during the course of a separation depending, for example, on thedesired cycle frequency. In certain examples, a square wave can beprovided by the function generator 3310 such that the solenoid valve3330 will cycle between an open and a closed position, with respect to agiven outlet. For example, where a 3-way solenoid valve is used, it canswitch the inlet flow between two different outlets and thus can be “on”with respect to one of the outlets and can be “off” with respect to theother outlet. Other waveforms, e.g., triangular, sinusoidal, sawtoothand the like, can also be used depending in the particular type ofswitching valve fluidically coupled to the wafer. In certainembodiments, each port of the microfluidic device may include anindividually controllable solenoid valve electrically coupled to acontroller and a gas source such that fluid flow through each port canbe individually controlled.

In certain embodiments, the microfluidic devices disclosed herein canpermit simultaneous analysis of two chromatograms. Referring to FIG. 36(FIGS. 34 and 35 are described below), a system 3600 is shown thatincludes a first injector 3610 fluidically coupled to a first column3615. The system 3600 also includes a second injector 3620 fluidicallycoupled to a second column 3625. Each of the first column 3615 and thesecond column 3625 are fluidically coupled to a microfluidic device3630. The microfluidic device 3630 is fluidically coupled to a vent 3640through a restrictor 3635. The microfluidic device 3630 is alsofluidically coupled to a MS detector 3650 through a restrictor 3645. TheMS detector 3650 is electrically coupled to a solenoid valve (not shown)between a gas source 3655 and the microfluidic device 3630. The MSdetector 3650 drives the midpoint solenoid valve modulation tosynchronize the microfluidic device 3630 with the scanning of the MSdetector 3650. Species eluting from both the first column 3615 and thesecond column 3625 can be directed to the MS detector 3650 using themicrofluidic device 3630. Such direction permits simultaneous processingof two chromatograms. The exact configuration of the microfluidic device3630 may be any of the illustrative configurations described herein orother suitable configurations.

In certain embodiments, a system configured to perform simultaneousconfirmatory chromatography is shown in FIG. 37. The system 3700includes an injector 3710 fluidically coupled to each of a first column3715 and a second column 3720. The injector 3710 splits the sample flowsuch that a portion is provided to the first column 3715 and a portionis provided to the second column 3720. Each of the first column 3715 andthe second column 3720 is fluidically coupled to a microfluidic device3725. The microfluidic device 3725 is fluidically coupled to a vent 3735through a restrictor 3730 and to a detector 3745 through a restrictor3740. If the separation media in the columns and column internaldiameters are the same, then the separation by each of the columnsshould be substantially the same. The switch 3725 can provide peaks fromboth the first column 3715 and the second column 3720 to the detector3745 such as, for example, a MS detector. The system can be ventedthrough the vent 3735 while the detector 3745 is kept at an operatingtemperature and pressure

In certain examples, an illustrative system configured formultidimensional separations and multiplexed detection is shown in FIG.38. The system 3800 includes an injector 3810 fluidically coupled to acolumn 3815 and a carrier gas source 3805. The column 3815 isfluidically coupled to a first microfluidic device 3820 which is coupledto a gas source 3822. The first microfluidic device 3820 is alsofluidically coupled to a second column 3825 and a second microfluidicdevice 3830. A restrictor 3827 is between the first microfluidic device3820 and the second microfluidic device 3830. The second microfluidicdevice 3830 is fluidically coupled to a gas source 3835. The secondmicrofluidic device 3830 is also fluidically coupled to a vent 3845 anda detector 3855. Peaks can elute from the first column 3815 and can beprovided to the second column 3825 or can be provided to the detector3855 or the vent 3845 using the first microfluidic device 3820 and thesecond microfluidic device 3830. Peaks may be selectively provided to adesired component based on the particular ports fluidically coupledwithin each of the first and second microfluidic devices 3820 and 3830,respectively. The first microfluidic device 3820 and second microfluidicdevice 3830 need not be the same but they may be the same. In someexamples, the first and second microfluidic devices are selected to bedifferent to provide for increased control of sample eluting from thecolumn or columns.

In certain embodiments and referring to FIG. 39, a dual column, singledetector configuration is shown. The system 3900 includes a first column3910 and a second column 3920 each fluidically coupled to a gas source3905 and 3915, respectively. Each of the first and second columns isalso fluidically coupled to a microfluidic device 3925. The microfluidicdevice 3925 includes a first buffer 3932 and a second buffer 3934. Theterm buffer is used interchangeably in certain instances with the termcharging chamber. The first buffer 3932 and the second buffer 3934 caneach be used to retain peaks from the columns. For example, species canelute from the column 3910 and be collected in the first buffer 3932.Once collected, the species can be directed to the detector 3945 througha restrictor 3940 using a switching gas from a gas source 3930 and aswitching valve 3927. Simultaneously, species eluting from the secondcolumn 3920 can be collected in the second buffer 3934. After thespecies from the first buffer 3932 have been provided to the detector3945, the valve 3927 can be switched such that the sample in the secondbuffer 3934 is now directed to the detector 3945 using the switchinggas. The capacity of the buffers 3932, 3934 is desirably matched and ofa sufficient volume for the column effluent flow rates and for themultiplexing frequency. For example, the buffers may each be from about80 microliters to about 150 microliters, for example about 120microliters, which is suitable for use with a column effluent rate ofabout 1 milliliter per minute at a multiplexing frequency of about 10Hz. Other suitable column effluent rates and multiplexing frequencieswill be selected by the person of ordinary skill in the art, given thebenefit of this disclosure.

In certain examples, a wafer that includes a buffer is shown in moredetail in FIG. 40. The wafer 4000 includes a plurality of ports and twobuffers. A port 4010 is fluidically coupled to a first buffer 4012 andto a second port 4015. The buffer 4012 is inline between the port 4010and a port 4020. A second buffer 4022 is inline between the ports 4025and 4030. The buffers 4012 and 4022 can be part of the microchannel butare larger in diameter. In particular, the diameters and lengths of thebuffers can be selected to be of sufficient capacity to accommodate theeffluent flowing from a column, at the applied mid-point pressure,during one cycle of the modulated switching valve. In certain examples,the length and width of the buffer can be selected so that the bufferfits inside the wafer and still provides a desired fluid capacity. Inoperation, the port 4010 can be fluidically coupled to a switching gassource, the port 4015 can be fluidically coupled to a first column toreceive effluent from the first column, the port 4020 can be fluidicallycoupled to a detector to provide sample to the detector, the port 4025can be fluidically coupled to another column to receive effluent fromthe second column, and the port 4030 can be fluidically coupled toanother switching gas source. As discussed in more detail below, thebuffers can be used along with flow control to reduce diffusionalbroadening of peaks.

In certain embodiments, the devices described herein can be used toprovide flow modulation. Flow modulation can provide substantialbenefits including improved peak detection. A typical chromatogram isshown in FIG. 41, in which the response is related to either the flowrate (for mass flow detectors) or the concentration (concentrationdependent detectors) of analytes flowing through a detector. When flowmodulation is used for the same sample used in FIG. 41, the signalshould appear closer to that shown in FIG. 42. The flow modulationallows the column effluent which is normally flowing at a slow rate(e.g. 1 mL/min) to flow into a chamber. For example, referring to FIG.43A, column effluent 4305 can flow from a column to a fluid flow path4310 and into a buffer or charging chamber 4320. When a switching valve4315 is in a first position, the column effluent 4305 will have atendency to build up in the charging chamber 4320, though some may exitthe charging chamber and be provided to a detector in the direction ofarrow 4325. Referring now to FIG. 43B, when the switching valve 4315 isactuated to a second position, a modulating gas will be provided in thedirection of arrow 4330. The flow rate of the modulating gas exceeds theflow rate of column effluent. This large difference in flow rate acts topush any column effluent 4305 in the charging chamber 4320 to thedetector in a single large pulse or bolus. This process results in alarge, narrow peak as shown in FIG. 42. In particular, this modulationhas the effect of introducing narrow bands of column effluent into thedetector separated by the clean modulating carrier gas as shown in FIG.42. The signal processing system desirably synchronizes discretedetector readings with the flow modulation so that these readings aretaken at the apexes of the pulses. Detector readings can also be takenin between the pulses to obtain a background signal. Several advantagescan be obtained using flow modulation optionally along with themicrofluidic devices described herein. For example, with mass-flowsensitive detectors, the mass flow of analyte may be increased by afactor of 50× or more giving rise to greater sensitivity and, improveddetection limits. With concentration dependent detectors, the modulationmay enable the column effluent to travel through the detector cell at ahigh rate but with little dilution. This result may enable relativelylarge cell detectors to be used with narrow bore columns without theloss in sensitivity normally associated with the use of a make-up gas.The ability to monitor the detector background signal between the pulsesmay help eliminate the effects of detector drift and improve detectionlimits and analytical stability. There may be opportunities to use amodulator with a conventional flame photometric detector to enabletime-gating of the optical emissions from the flame subsequent to eachpulse to improve selectivity and reduce noise—in a similar way asachieved using, for example, certain pulsed flame photometric detectors.Also, a good flow modulator may form the basis of a GCxGC system.

In certain embodiments, the charging chamber shown in FIGS. 43A and 43Bmay have some limitations. In FIG. 43A, the switching valve preventsflow of a modulating gas into the charging chamber 4320 and so the flowrate of gas into the detector will be that of the column effluent. Whenthe switching valve is actuated to a second position, there is a suddenincrease in the flow rate entering the detector. This slow-fast-slowflow rate of carrier gas can lead to noise and instability in thedetector. If such noise is present, a 3-way switching valve can be usedas shown in FIGS. 44A and 44B to provide a more stable flow rate of gasinto the detector. Referring to FIGS. 44A and 44B, a device includes acharging chamber 4420 fluidically coupled to a column (not shown)through a fluid flow path 4405. The device also includes a fluid flowpath 4410 between a 3-way switching valve 4415 and the charging chamber4420. A by-pass flow path 4430 is also fluidically coupled to the 3-wayswitching valve 4415. When the 3-way switching valve 4415 fluidicallycouples a modulating gas from fluid flow path 4435 to the by-pass flowpath 4430 (FIG. 44A), modulating gas travels into the by-pass flow pathand out to a detector through fluid flow path 4425. In this state,effluent from the column can build up in the charging chamber 4420. Whenthe 3-way switching valve is actuated to a different position (FIG.44B), the modulating gas can be provided to the charging chamber 4420through the fluid flow path 4410 and can act to force the accumulatedeffluent from the charging chamber to the detector along the fluid flowpath 4425. One cycle of the 3-way switching valve will produce one pulseinto the detector. Thus to generate, for example, fifty pulses eachsecond, the 3-way switching valve must oscillate at 50 Hz. This highlevel of cycling can place significant stress on the switching valve. Inaddition, while the chamber is being flushed as shown in FIGS. 43B and44B, column effluent will continue to enter the chamber. This materialwill be diluted and effectively lost from the analysis.

In certain embodiments, more than one charging chamber can be used inthe flow modulation methods described herein and the microfluidicdevices described herein. An illustration of this configuration is shownin FIGS. 45A and 45B. The device includes a 3-way switching valve 4515fluidically coupled to a modulating gas through a fluid flow path 4510.The switching valve 4515 is fluidically coupled to a first chamber 4525through a fluid flow path 4520 and to a second chamber 4540 through afluid flow path 4535. A column (not shown) is fluidically coupled toeach of the first chamber 4525 and the second chamber 4540 through aninlet 4505. The chamber 4525 is fluidically coupled to a detector (notshown) through fluid flow paths 4530 and 4550, and the chamber 4540 isfluidically coupled to the detector through fluid flow paths 4545 and4550. While one of the chambers 4525, 4540 is being charged, the otherchamber is being flushed with the modulating gas. This arrangementshould generate two pulses for each cycle of the switching valve, whichpermits the switching valve to oscillate at half the speed as the singlechamber design to achieve the same performance. In addition, none of thecolumn effluent is wasted—it is always charging one of the chambers. Asmall flow of the modulating gas between the chambers can prevent orreduce diffusion of sample vapor into the chamber being swept. The flowrate to the detector will be the same in either position of theswitching valve.

In certain examples, the microfluidic devices described herein can beconfigured with one or more of the charging chambers. FIG. 46 shows oneillustration of a microfluidic device with at least one chargingchamber. The microfluidic device 4600 includes a first chamber 4610 anda second chamber 4615. A column effluent port 4640 is fluidicallycoupled to each of the first chamber 4610 and the second chamber 4615.Modulating gas may be introduced at a port 4630 or a port 4650 dependingon which of the chambers 4610, 4615 is swept or flushed. A switchingvalve (not shown) is fluidically coupled to each of the ports 4630 and4650 to control which port receives the modulating gas. One or morerestrictors may be present to balance the flow in the system. When themodulating gas is introduced into the port 4630 the modulating gas willsweep chamber 4615 into the detector, and the column effluent will becharging the chamber 4610. When the modulating gas is switched to theport 4650, the chamber 4610 will be swept and the chamber 4610 will becharged. The chambers can be designed to be long and narrow to minimizedilution and dispersion of the sample as it is flushed.

In certain embodiments where a charging chamber is present, the chambergeometry can be selected to suit the operating conditions. For example,the following variables can be considered when selecting the chambergeometry and dimensions: column flow rate, modulating gas flow rate,pressure inside the microfluidic device and switching valve modulationfrequency. In one illustration, if the column flow rate is in the range0.5 to 3 mL/min (e.g., columns with a maximum internal diameter of 0.32mm) and the flow rate into the detector is about 50 mL/min, then theseassumptions provide a compression factor in the range 17× to 100×. Ifthe internal pressure of the microfluidic device is about 8 psig, then apiece of fused silica tubing can be connected to the detector to provideabout 50 mL/min at 8 psig. The restrictor geometry will be dependent onthe particular detector selected. The charging chamber is desirablylarge enough to hold all the column effluent eluting from the columnbefore it is pulsed. At the pressure inside the microfluidic device, themaximum volumetric flow rate from the column will be3×(Ambient Pressure)/(Microfluidic device Pressure+Ambient Pressure)=33315/23=˜2 mL/minTable II lists the chamber capacities desired for a range of switchingvalve modulation frequencies with a 2 mL/min flow rate at 8 psig.

TABLE II Predicted chamber capacity requirements Charge Time ValveFrequency (Hz) (milliseconds) Chamber Capacity (μL) 5 100 13.33 10 506.67 20 25 3.33 50 10 1.33If the channels of the microfluidic device are 80 microns in height andthe chamber length is about 30 mm, the chamber widths can be selected asshown in Table III.

TABLE III Predicted chamber dimensions Chamber Chamber Chamber ChamberCapacity Height Length Width (μL) (μm) (mm) (μm) 13.33 80 30 444 6.67 8030 222 3.33 80 30 111 1.33 80 30 44Tables II and III are provided as a guide, but any of the assumptionscan be varied which would change the exact dimensions selected.

In certain embodiments, the internal chamber channel geometry can alsobe selected to provide desired flow properties. For example, thegeometry of the microchannels between the column port and each of thetwo chambers can alter the fluid flow. If these are too wide, themodulating gas will be able to cross between the two chambers and flushthem both simultaneously. If they are too narrow, then the column portwill increase in pressure from the column effluent and so the effluentwill split into both chambers. The flow of modulating gas into thechamber being charged should be kept very low (e.g., <50 μL/min). FIG.47 shows one illustration of how this process can be controlled using amicrofluidic device. The microfluidic device 4700 includes a firstchamber 4710 and a second chamber 4715. The microfluidic device 4700also includes a plurality of ports. A column effluent port 4740 isfluidically coupled to the first chamber 4710 and the second chamber4715. A 3-way switching valve (not shown) is fluidically coupled to aport 4730 and 4750 and can provide a modulating gas to either of theports 4730, 4750 depending on the position of the valve. If themodulating gas is switched to the port 4730, then 50 mL/min of gasshould flow through the chamber 4715 and out to a detector through afluid flow path connected to port 4720. The chamber 4715 should notimpose any significant restriction but the fluid flow path 4755 to theport 4720 may provide restricted flow. The flow rate of the modulatinggas from the port 4730 through the chamber 4710 will be controlled by afluid flow path 4760. It is the relative impedance of the flow paths4755 and 4760 that will dictate the flow rate of the modulating gasthrough the chamber 4710 and the chamber 4715. Thus, the dimensions ofthe fluid flow paths 4755 and 4760 can be restricted or expanded,relative to the dimensions of other portions of the microchannel, toprovide a desired flow rate through the microfluidic device.

In certain embodiments, the microfluidic devices described herein can beused, for example, to split peaks. For example, individual peaks can becut and provided to different detectors (or different components) or asingle peak may be split and provided to two different components. Forexample, where a particular species in the sample is highlyconcentrated, it may be desirable to split that sample peak and send aportion of it to a vent rather than send the entire peak to a detector.Such splitting can overcome the dynamic range limitations of a columnand/or a detector. In addition, large injection volumes can be used andthe solvent peak can be split (or removed) entirely to avoid overloadingthe detector. One configuration of a system that is configured for peaksplitting is shown in FIG. 48. The system 4800 includes an injector 4810fluidically coupled to a carrier gas source 4805 and a first column4815. The first column 4815 is fluidically coupled to a microfluidicdevice 4820, which itself is fluidically coupled to a switching valve4825. A second column 4830 is fluidically coupled to the microfluidicdevice 4820 and a detector 4835. The microfluidic device 4820 is alsofluidically coupled to a restrictor 4840 and a vent 4845. Depending onthe position of the switching valve 4845, peaks eluting from the firstcolumn 4815 can be cut and provided to the second column 4830 or to thevent 4845. For example, contamination peaks or solvent peaks can beselectively provided to the vent 4845 so that they do not interfere withdetection of sample peaks, which can be provided to the second column4830 and to the detector 4835. In some examples, a portion of a peak canbe cut and provided to the vent. For example, where one component in asample (or a contaminant in a sample) is present at a substantiallyhigher concentration than the other components, the highly concentratedcomponent may be present at a concentration higher than the dynamicrange of the detector, which can result in a flat top peak if the signalexceeds the maximum detector signal. By splitting the peak into two ormore portions, the concentration may fall within the detector range toprovide a more accurate assessment of how much of that component ispresent in the sample. Different speaks can be split different amounts,e.g., 25%, 50%, 75% or other splitting percentages. The system of FIG.48 also permits venting through the vent 4845. Such venting can overcomeissues resulting from large solvent amounts, which can permit largerinjection volumes to be used. In addition, backflushing and MS ventfunctionalities can be performed as described herein.

In certain embodiments, a system can be configured to split differentpeaks or provide different peaks to two or more detectors. Referring toFIG. 49, a system 4900 includes an injector 4905 fluidically coupled toa carrier gas source 4910 and a column 4915. The column 4915 isfluidically coupled to a microfluidic device 4975. A midpoint pressureregulator 4920 may optionally be fluidically coupled to the microfluidicdevice 4975. The microfluidic device 4975 is fluidically coupled to afirst detector 4940 through a restrictor 4925, fluidically coupled to asecond detector 4945 through a restrictor 4930, and fluidically coupledto a MS device 4950 through a restrictor 4935. In operation of thesystem, peaks may be cut and a portion can be provided to the firstdetector 4940 and the remainder of the cut peak can be provided to thesecond detector 4945. In an alternative, a portion of the peak can beprovided to the MS device 4950. In some embodiments, entire peaks may beprovided to the different detectors. Other uses of the system 4900 willbe readily selected by the person of ordinary skill in the art, giventhe benefit of this disclosure.

In certain embodiments, the sample can be split prior to any separation.One configuration of such a system is shown in FIG. 50. The system 5000includes an injector 5005 fluidically coupled to a carrier gas source5010. The injector 5005 is fluidically coupled to a microfluidic device5075. A midpoint pressure regulator 5020 may optionally be fluidicallycoupled to the microfluidic device 5075 through a restrictor 5015. Aswitching gas source 5020 can be fluidically coupled to the microfluidicdevice 5075. The microfluidic device 5075 is fluidically coupled to afirst detector 5040 through a first column 5025, fluidically coupled toa second detector 5045 through a second column 5030, and fluidicallycoupled to a MS device 5050 through a third column 5035. Sample can beinjected into the system using the injector 5005 and can be split to thedifferent components using the microfluidic device 5075. Separation canbe performed using the columns 5025, 5030 and 5035 and the peaks can beprovided to the corresponding detector. The configuration shown in FIG.50 permits simultaneous analysis of a sample using different types ofdetectors and/or different types of column materials.

In certain embodiments, splitting of the peaks can permit use ofdifferent carrier gases. One configuration of such a system is shown inFIG. 51. The system 5100 includes an injector 5105 fluidically coupledto a first carrier gas source 5110. The injector 5105 is fluidicallycoupled to a first column 5115. A switching valve 5125 can befluidically coupled to a microfluidic device 5155, a splitter 5160 and asecond carrier gas source 5120, which may be the same as the firstcarrier gas source or may be different. The microfluidic device 5155 isalso fluidically coupled to a third gas source 5145, which may be thesame or may be different from the first and second carrier gas sources5110, 5120. The microfluidic device 5155 is further fluidically coupledto a first detector 5135 through a second column 5140 and to a thirddetector 5165 through a third column 5150. In one scheme using thesystem of FIG. 51, the first gas source 5110 can be nitrogen which isused at a flow rate of 10 cm/sec. The second gas source 5120 may also benitrogen, which can be introduced at a sufficient flow rate to provide aflow rate through the second column 5140 of about 40 cm/sec. The secondgas source can be provided, for example, to sweep effluent from acharging chamber 5130. The third gas source can be hydrogen and can beprovided at a flow rate of about 40 cm/sec to the third column 5150. Inthis configuration, the different carrier gases can provide differentseparation using the second and third columns 5140, 5150. Such differentcarrier gases may be desirable where, for example, a single type ofcarrier gas does not provide suitable separation of all the componentsin the sample.

In certain embodiments, the systems described herein can be used formultidimensional separations. One illustration is shown in FIGS. 52A and52B. The system 5200 includes an injector 5210 fluidically coupled to acarrier gas source 5205 and a first column 5215. The first column 5215is fluidically coupled to a microfluidic device 5220, which itself isfluidically coupled to a modulating gas source 5225. The microfluidicdevice 5220 is also fluidically coupled to a first detector 5235 througha restrictor 5230 and to a second detector 5245 through a second column5240. The microfluidic device 5220 is also typically in fluidcommunication with a switching valve (not shown), which can permit fluidflow from the first column 5215 to the second column 5240 and seconddetector 5245 in one position (FIG. 52A) and can permit fluid flow tothe second detector 5235 through the restrictor 5230 in another position(FIG. 52B). The position of the switching valve may be changed tocontrol which components of the system receive column effluent. Suchdirection of flow provides for different data sets which can be used toprovide a better analysis of components in the sample and can be, ifdesired, provided on the same chromatogram for easier analysis.

In some embodiments, the multidimensional separation can occur aftercolumn effluent is split but before any separation has occurred. Oneconfiguration of a system that uses a split flow for multidimensionalanalysis is shown in FIG. 53. The system 5300 includes an injector 5310fluidically coupled to a carrier gas source 5305 and a restrictor 5315.The restrictor 5315 is fluidically coupled to a microfluidic device5320, which itself is fluidically coupled to a modulating gas source5325. The microfluidic device 5320 is also fluidically coupled to afirst detector 5335 through a first column 5330 and to a second detector5345 through a second column 5340. The microfluidic device 5320 is alsotypically in fluid communication with a switching valve (not shown),which can split the fluid flow from the injector 5310 and provide thesplit flow to the different columns of the system 5300. Such splittingpermits the use of different separation media in the two columns toprovide different data sets and different separations using a singlesystem.

In certain examples, a system for use in a multidimensional separation,e.g., GCxGC, can include three or more columns. One system that includesthree columns is shown in FIG. 54. The system 5400 includes an injector5410 fluidically coupled to a carrier gas source 5405 and a first column5415. The first column 5415 is fluidically coupled to a microfluidicdevice 5420, which itself is fluidically coupled to a modulating gassource 5425. The microfluidic device 5420 is also fluidically coupled toa first detector 5435 through a second column 5430 and to a seconddetector 5445 through a third column 5440. The microfluidic device 5420is also typically in fluid communication with a switching valve (notshown), which can split the column effluent flow (or particular peaks ifdesired) from the first column 5415 and provide the split flow to thetwo other columns of the system 5400. Such splitting permits the use ofdifferent separation media in the three columns, if desired, to providedifferent data sets and different separations using a single system.

In certain examples, the devices, methods and systems described herein(or portions thereof) can be implemented or controlled using a computeror other device that includes a processor, or the devices and systemsdescribed herein can be electrically coupled to a computer system orprocessor. Such computer implemented methods can provide for more userfriendly implementation of the methods by permitting control using agraphical user interface or the like. In addition, the computer can beused to monitor flow rates, receive data from one or more detectors andto store or recall separation routines for subsequent use. The computersystem typically includes at least one processor optionally electricallycoupled to one or more memory units. The computer system may be, forexample, a general-purpose computer such as those based on Unix, IntelPENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, or any other type of processor. Insome examples, the processor may be an inexpensive processor that may beprogrammable to receive inputs and output treatment parameters based onthe received inputs. It should be appreciated that one or more of anytype computer system may be used according to various embodiments of thetechnology. Further, the system may be located on a single computer ormay be distributed among a plurality of computers attached by acommunications network. A general-purpose computer system may beconfigured, for example, to perform any of the described functionsincluding but not limited to: restrictor length and diametercalculations, gas source control, switching valve control, temperaturecontrol, run times, and the like. It should be appreciated that thesystem may perform other functions, including network communication, andthe technology is not limited to having any particular function or setof functions.

For example, various aspects may be implemented as specialized softwareexecuting in a general-purpose computer system. The computer system mayinclude a processor connected to one or more memory devices, such as adisk drive, memory, or other device for storing data. Memory istypically used for storing programs and data during operation of thecomputer system. Components of the computer system may be coupled by aninterconnection device, which may include one or more buses (e.g.,between components that are integrated within a same machine) and/or anetwork (e.g., between components that reside on separate discretemachines). The interconnection device provides for communications (e.g.,signals, data, instructions) to be exchanged between components of thesystem. The computer system typically is electrically coupled to thedetector such that electrical signals may be provided to and from thedetector to the computer to receive data for storage and/or processing.The computer system may also include one or more input devices, forexample, a keyboard, mouse, trackball, microphone, touch screen, manualswitch (e.g., override switch) and one or more output devices, forexample, a printing device, display screen, speaker. In addition, thecomputer system may contain one or more interfaces (not shown) thatconnect the computer system to a communication network (in addition oras an alternative to the interconnection device).

The storage system typically includes a computer readable and writeablenonvolatile recording medium in which signals are stored that define aprogram to be executed by the processor or information stored on or inthe medium to be processed by the program. For example, the oventemperatures, flow rates, switching valve position and modulationfrequencies and the like for a particular separation may be stored onthe medium. The medium may, for example, be a disk or flash memory.Typically, in operation, the processor causes data to be read from thenonvolatile recording medium into another memory that allows for fasteraccess to the information by the processor than does the medium. Thismemory is typically a volatile, random access memory such as a dynamicrandom access memory (DRAM) or static memory (SRAM). It may be locatedin the storage system or in the memory system. The processor generallymanipulates the data within the integrated circuit memory and thencopies the data to the medium after processing is completed. A varietyof mechanisms are known for managing data movement between the mediumand the integrated circuit memory element and the technology is notlimited thereto. The technology is also not limited to a particularmemory system or storage system.

In certain examples, the computer system may also includespecially-programmed, special-purpose hardware, for example, anapplication-specific integrated circuit (ASIC). Aspects of thetechnology may be implemented in software, hardware or firmware, or anycombination thereof. Further, such methods, acts, systems, systemelements and components thereof may be implemented as part of thecomputer system described above or as an independent component.

Although a computer system is described by way of example as one type ofcomputer system upon which various aspects of the technology may bepracticed, it should be appreciated that aspects are not limited tobeing implemented on the illustrated computer system. Various aspectsmay be practiced on one or more computers having a differentarchitecture or components. The computer system may be a general-purposecomputer system that is programmable using a high-level computerprogramming language. The computer system may be also implemented usingspecially programmed, special purpose hardware. In the computer system,the processor is typically a commercially available processor such asthe well-known Pentium class processor available from the IntelCorporation. Many other processors are available. Such a processorusually executes an operating system which may be, for example, theWindows 95, Windows 98, Windows NT, Windows 2000 (Windows ME), WindowsXP or Windows Vista operating systems available from the MicrosoftCorporation, MAC OS System X operating system available from AppleComputer, the Solaris operating system available from Sun Microsystems,or UNIX or Linux operating systems available from various sources. Manyother operating systems may be used, and in certain embodiments a simpleset of commands or instructions may function as the operating system.

In accordance with certain examples, the processor and operating systemmay together define a computer platform for which application programsin high-level programming languages may be written. It should beunderstood that the technology is not limited to a particular computersystem platform, processor, operating system, or network. Also, itshould be apparent to those skilled in the art, given the benefit ofthis disclosure, that the present technology is not limited to aspecific programming language or computer system. Further, it should beappreciated that other appropriate programming languages and otherappropriate computer systems could also be used. In certain examples,the hardware or software is configured to implement cognitivearchitecture, neural networks or other suitable implementations.

One or more portions of the computer system may be distributed acrossone or more computer systems coupled to a communications network. Thesecomputer systems also may be general-purpose computer systems. Forexample, various aspects may be distributed among one or more computersystems configured to provide a service (e.g., servers) to one or moreclient computers, or to perform an overall task as part of a distributedsystem. For example, various aspects may be performed on a client-serveror multi-tier system that includes components distributed among one ormore server systems that perform various functions according to variousembodiments. These components may be executable, intermediate (e.g., IL)or interpreted (e.g., Java) code which communicate over a communicationnetwork (e.g., the Internet) using a communication protocol (e.g.,TCP/IP). It should also be appreciated that the technology is notlimited to executing on any particular system or group of systems. Also,it should be appreciated that the technology is not limited to anyparticular distributed architecture, network, or communication protocol.

In accordance with certain examples, various embodiments may beprogrammed using an object-oriented programming language, such asSmallTalk, Basic, Java, C++, Ada, or C# (C-Sharp). Other object-orientedprogramming languages may also be used. Alternatively, functional,scripting, and/or logical programming languages may be used. Variousconfigurations may be implemented in a non-programmed environment (e.g.,documents created in HTML, XML or other format that, when viewed in awindow of a browser program, render aspects of a graphical-userinterface (GUI) or perform other functions). Certain configurations maybe implemented as programmed or non-programmed elements, or anycombination thereof.

In certain examples, a user interface may be provided such that a usermay enter desired flow rates, tubing lengths and diameters, columntypes, solvent gradient runs and other information commonly enteredprior to a gas or liquid chromatography separation is commenced. Otherfeatures for inclusion in a user interface will be readily selected bythe person of ordinary skill in the art, given the benefit of thisdisclosure.

In certain embodiments, the microfluidic devices described herein may bepackaged in a kit optionally with instructions for using themicrofluidic device. In some examples, the kit may further include acomputer readable medium that contains algorithms suitable forimplementing flow control or modulation as described herein. The kit mayfurther include fittings, tubing, restrictors or the like of a desiredlength or diameter to facilitate a desired flow rate in the system. Insome examples, one or more separation columns may also be included inthe kit.

Certain specific examples are described below to illustrate further someof the new and useful features of the technology described herein.

Example 1

To validate the tubing diameter algorithms, a length of fused silicatubing (listed as having an internal diameter of 150 microns) was testedwith helium and nitrogen carrier gases. A least squares linear fit wasapplied to the flow rate versus the square of the absolute appliedpressure to establish the value of the constant b in Equation (17) andd_(c) was calculated from Equation (18). The ambient pressure wasdetermined from a digital barometer at the location and the viscosity atthe ambient temperature was taken from tables. The results are given inTables IV (helium gas) and V (nitrogen gas) and are listed in order ofdecrementing length L.

TABLE IV Measured Flow Rate (mL/min) L 80 70 60 50 40 30 20 10 5 Fitd_(c) (cm) (psig) (psig) (psig) (psig) (psig) (psig) (psig) (psig)(psig) r² (μm) 200 42.40 33.70 25.90 19.30 13.50 8.74 4.68 1.87 1.0000152.9 190 44.20 35.40 27.10 20.30 14.30 9.16 4.91 2.01 0.9999 152.6 18046.90 37.40 28.70 21.50 15.10 9.64 5.20 2.15 1.0000 152.8 170 49.3039.00 30.30 22.60 15.90 10.10 5.48 2.29 0.9999 152.4 160 53.10 42.3032.60 24.20 16.90 10.80 5.87 2.44 1.0000 153.1 150 55.50 44.30 34.4025.50 18.00 11.40 6.25 2.61 1.0000 152.3 140 60.70 48.40 37.20 27.4019.50 12.50 6.82 2.84 1.0000 153.0 130 65.40 52.10 40.00 29.60 21.0013.50 7.31 3.04 1.0000 153.0 120 70.50 56.40 43.60 32.30 22.60 14.608.01 3.20 1.0000 152.9 110 76.30 61.20 47.40 35.10 24.60 15.90 8.75 3.561.0000 152.6 100 84.00 67.00 52.00 38.40 27.00 17.40 9.60 3.86 1.0000152.6 90 93.00 74.20 57.80 42.80 29.80 19.40 10.60 4.25 1.90 1.0000152.5 80 104.00 82.90 64.70 48.00 33.60 21.60 12.00 4.65 2.10 0.9999152.3 70 118.00 94.10 73.20 54.40 38.20 24.60 13.80 5.28 2.35 0.9999152.0 60 136.00 109.00 84.80 63.10 44.20 28.30 15.90 6.16 2.74 1.0000151.6 50 163.00 130.00 101.00 75.00 52.80 34.00 19.00 7.42 3.30 1.0000151.4 40 201.00 162.00 125.00 93.60 66.10 42.70 23.70 9.48 4.14 0.9999151.0 30 265.00 211.00 166.00 123.00 87.10 56.50 31.50 12.60 5.43 0.9998150.4

TABLE V Measured Flow Rate (mL/min) L 80 70 60 50 40 30 20 10 5 Fitd_(c) (cm) (psig) (psig) (psig) (psig) (psig) (psig) (psig) (psig)(psig) r² (μm) 200 37.60 28.90 21.70 15.30 9.77 5.19 2.21 0.9999 153.5190 38.70 30.00 22.40 15.80 10.20 5.47 2.25 0.9999 152.7 180 40.30 31.5023.50 16.50 10.60 5.74 2.37 0.9999 152.3 170 43.80 33.80 25.20 17.8011.40 6.19 2.55 1.0000 153.1 160 46.30 35.90 26.90 18.90 12.10 6.64 2.740.9999 153.0 150 47.60 37.20 27.80 19.90 12.80 7.02 2.90 0.9998 151.5140 52.70 41.20 30.40 21.50 14.00 7.64 3.11 0.9999 152.8 130 56.10 44.0032.80 23.20 15.10 8.23 3.37 0.9998 152.4 120 59.60 47.00 35.00 24.8016.10 8.80 3.60 0.9998 151.7 110 52.10 38.40 27.10 17.50 9.76 3.941.0000 152.9 100 56.90 42.50 29.70 19.30 10.70 4.26 1.0000 152.8Tables IV and V show that a highly consistent value for the internaldiameter is achieved as the restrictor tubing is progressivelyshortened. The mean values (152.3 μm for helium and 152.6 μm fornitrogen) are very close and the precision in the calculations isexcellent (0.49% RSD for helium and 0.40% RSD for nitrogen).

To validate the accuracy of these results, the sections of fused silicatubing removed during the flow measurement tests were examined under a500× magnification microscope and the true diameters determined byphotomicrography. FIG. 55 shows a photomicrograph of the end of onesection of the fused silica tubing with an overlaid graticule with 10microns scale divisions. Table VI lists the results of the manualmeasurements taken through the microscope. These measurements agree veryclosely with the calculated values in Tables IV and V.

TABLE VI Section Taken (cm) Measured Bore (μm) 200 153 180 153 160 152140 152 120 153 100 153Thus, to better determine the true size of tubing used in fluidchromatography systems, a calibration protocol can be implemented toaccurately assess the true internal diameter of tubing, e.g., columns,restrictors, etc. used in the systems.

Example 2

The results from a thermal desorption system that uses algorithms basedon the equations described above to control the flow rate of gas througha transfer line and a column are shown in FIG. 56. The carrier gas wasdoped with a fixed concentration of methane so that the mass flow rateof gas eluting from the column could be directly monitored by a flameionization detector. FIG. 56 shows plots of the detector signal during acolumn temperature program with constant pressure control and then withconstant flow control. As can be seen from these plots, the mass flowrate is reasonably constant during the program when constant flowcontrol is used.

In FIG. 56, the flow rates were controlled as the column temperature wasincreased. The methane was added to the column and the resulting signalwas used to monitor the detector response as a function of the flowrate. The oven was heated to 40° C. and maintained at this temperaturefor 1 minute. The temperature was then increased by 10° C./minute up toa final temperature of 300° C. The final temperature was maintained for10 minutes. Curve 5610 represents the actual flow rate, curve 5620represents the expected flow rate, and curve 5630 represents the flowrate without use of the flow control (where constant pressure controlwas used). Where pressure control was used, the flow rate differedmarkedly from the desired flow rate. Where the flow control algorithmsdescribed herein are implemented, the flow rate closely tracked that ofthe desired flow rate.

Example 3

FIGS. 34 and 35A-35C show illustrations of chromatography peaks wheremodulation was performed, as described herein. Referring to FIG. 34, asingle peak is shown. The modulation breaks the peak into a plurality ofindividual modulated portions. Modulation was performed at 10 Hz usingthe controller of FIGS. 33A and 33B and the microfluidic device of FIG.11. The column used was a 30 m×0.25 mm×0.25 micron methyl siliconecolumn. Helium was used as the mobile phase. The inlet pressure was 40psig, and the midpoint pressure was 30 psig (helium gas). The oven washeated to 35° C. and maintained at this temperature for 1 minute. Thetemperature was then increased by 10° C. per minute up to a temperatureof 300° C. A fast FID detector at 275° C. was used to detect the peaks.A 100 mL/min split injector was used to introduce the sample, which was1 microgram/microliter NIOSH aromatics (0.2 microliters was injected).

Referring to FIGS. 35A-35C, three different traces are shown. In FIG.35A, two peaks 3510, 3520 representative of sample effluent from a firstcolumn are shown. In FIG. 35B, a single peak 3530 representative ofsample effluent from a second column is shown. These peaks can beanalyzed simultaneously using the modulation techniques describedherein. Referring to FIG. 35C, the modulated output of the differentsamples is shown where, for example, the sample from the first andsecond columns can be provided to a single output. The peaks 3510 and3530 overlap or are interlaced in the modulated output as shown in themodulated signal group 3540. The peak 3520 is shown as a modulated peak3550. In this manner, sample peaks from different columns can beanalyzed simultaneously.

Example 4

A microfluidic device that included an internal bypass restrictor isshown in FIG. 57. The resulting microfluidic device (FIG. 57) included aplurality of ports 5705, 5710, 5715, 5720, 5725 and 5730. The port 5705is designed to receive effluent from a column. A switching gas from asolenoid valve can be connected to each of the ports 5715 and 5725.Outlet ports 5710 and 5730 can be connected to columns or restrictors orother devices. The internal bypass restrictor 5750 has a diameter thatis less than that of other portions 5760 of the internal microfluidicchannel. The particular diameter and length selected for this internalbypass restrictor can provide for flow control using the microfluidicdevice.

When introducing elements of the examples disclosed herein, the articles“a,” “an,” and “the” are intended to mean that there are one or more ofthe elements. The terms “comprising,” “including” and “having” areintended to be open ended and mean that there may be additional elementsother than the listed elements. It will be recognized by the person ofordinary skill in the art, given the benefit of this disclosure, thatvarious components of the examples can be interchanged or substitutedwith various components in other examples.

Although certain features, aspects, examples and embodiments have beendescribed above, additions, substitutions, modifications, andalterations of the disclosed illustrative features, aspects, examplesand embodiments will be readily recognized by the person of ordinaryskill in the art, given the benefit of this disclosure.

What is claimed is:
 1. A system for modulating flow of a fluid in achromatography system, the system comprising: a microfluidic devicefluidically coupled to a switching valve, in which the microfluidicdevice comprises a first charging chamber and a second charging chamber;and a detector fluidically coupled to a first outlet of the microfluidicdevice, wherein the switching valve is configured to actuate between afirst position and a second position, wherein the first position isconfigured to permit fluid flow from a modulating gas source to thefirst charging chamber of the microfluidic device to provide columneffluent from the first charging chamber to the detector fluidicallycoupled to the microfluidic device, and wherein the second position isconfigured to permit fluid flow from the modulating gas source to thesecond charging chamber of the microfluidic device to provide columneffluent from the second charging chamber to the detector fluidicallycoupled to the microfluidic device.
 2. The system of claim 1, in whichthe switching valve is a 3-way solenoid valve that is actuated at afrequency of about 10 Hz to about 100 Hz.
 3. The system of claim 1,further comprising a restrictor between the first outlet of themicrofluidic device and the detector, in which the restrictor isconfigured to balance pressure in system.
 4. The system of claim 3, inwhich a restrictor length of the restrictor is calculated using thefollowing equation$L_{r} = {\frac{\left\lbrack {{\frac{\pi \times T_{a} \times d_{r}^{4}}{256 \times F_{a} \times p_{a}} \times \left( {p_{i}^{2} - p_{or}^{2}} \right)} - {T_{r\; 2} \times \eta_{r\; 2} \times L_{r\; 2}}} \right\rbrack}{T_{r\; 1} \times \eta_{r\; 1}} + L_{r\; 2}}$where L_(r1) is length of the restrictor inside the oven, L_(r2) islength of the restrictor inside the detector, η_(r1) is viscosity ofcarrier gas at temperature of an oven, η_(r2) is viscosity of carriergas at a detector temperature, T_(r1) is absolute temperature of theoven, T_(r2) is the absolute temperature of the detector, p_(i) is inletpressure, p_(or) is outlet pressure and F_(a) is flow rate.
 5. Thesystem of claim 1, further comprising a second detector fluidicallycoupled to a second outlet of the microfluidic device.
 6. The system ofclaim 5, further comprising a first restrictor between the first outletand the detector and a second restrictor between second outlet and thesecond detector.
 7. The system of claim 1, further comprising aninternal restrictor within the microfluidic device.
 8. The system ofclaim 7, in which an internal diameter of the internal restrictor iscalculated using the following equation$d_{r} = \sqrt[4]{\frac{256 \cdot L \cdot p_{a} \cdot \eta}{b \cdot \pi}}$where d_(r) is the internal diameter of the restrictor, L is restrictorlength, p_(a) is absolute pressure, η is viscosity of a carrier gas andb is a constant.
 9. The system of claim 1, in which the microfluidicdevice comprises an internal microchannel comprising a variable diameterat different portions of the internal microchannel.
 10. The system ofclaim 1, in which the switching valve comprises a laminated membrane.11. The system of claim 1, in which the switching valve is a 3-waysolenoid valve and the microfluidic device is configured as a laminatedwafer comprising stainless steel plates.
 12. The system of claim 1, inwhich the microfluidic device is configured as a laminated wafer. 13.The system of claim 12, in which the first charging chamber and thesecond charging chamber are formed from laminating different layers ofthe laminated wafer to each other.
 14. The system of claim 12, in whichthe laminated wafer comprises stainless steel plates.
 15. The system ofclaim 14, in which the laminated wafer comprises a plurality of portseach connected to each other through an internal microfluidic channel.16. The system of claim 15, in which the internal microfluidic channelcomprises an internal bypass restrictor.